Compositions, kits and related methods for the detection and/or monitoring of salmonella

ABSTRACT

Provided are compositions, kits, and methods for the identification of Salmonella. In certain aspects and embodiments, the compositions, kits, and methods may provide improvements in relation to specificity, sensitivity, and speed of detection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 14/215,407,filed Mar. 17, 2014, which is a continuation of application Ser. No.12/995,168, having a 35 U.S.C. § 371(c) date of Nov. 29, 2010, now U.S.Pat. No. 8,703,421, which is a U.S. national phase application ofInternational Application No. PCT/US2009/045738, filed May 29, 2009,which claims the benefit of priority under 35 U.S.C. § 119(e) to U.S.provisional Application No. 61/057,787, filed May 30, 2008, the entirecontents of each of which are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicformat. The Sequence Listing is provided as a file entitled“2019-01-09_01158-0018-02US_Seq_List_ST25.txt” created on Jan. 9, 2019,which is 43.7 KB in size. The information in the electronic format ofthe sequence listing is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

The Salmonella genus is part of a large family of gram-negative bacteriafound in humans, animals, foods and the environment. Salmonella is arod-shaped enterobacteria that infects humans and causes typhoid fever,paratyphoid fever, and food-borne illness. In the U.S., it is the mostfrequently reported food-borne disease. Worldwide, it is the second mostcommon food-borne illness in the world. Salmonellosis affects 100 to 300per 100,000 population. In 2005, there were ˜36,000 cases reported tothe CDC. Salmonellosis is transmitted by the ingestion of food derivedfrom infected animals or contaminated by feces (animal or human).Symptoms include acute enterocolitis with sudden onset of headache,abdominal pain, diarrhea, nausea and sometimes vomiting. Incubation timeis between 6 to 72 h (average of 12-36 h). Enteric bacteria have complexgenotypic and phenotypic characters that are oftentimes shared acrossgenera. As a result, their genomes share many common sequences. Thereremains a need in the art for a rapid and robust detection system thatcan specifically and selectively identify Salmonella in a sample ofinterest.

SUMMARY OF THE INVENTION

The present invention relates to compositions, kits, and methods used inthe detection of Salmonella. The invention is based at least in part onthe discovery that certain Salmonella sequences are surprisinglyefficacious for the detection of Salmonella. In certain aspects andembodiments, particular regions of the Salmonella 23S rRNA have beenidentified as preferred targets for nucleic acid amplification reactionswhich provide improvements in relation to specificity, sensitivity, orspeed of detection as well as other advantages.

Therefore, according to one aspect, there are provided compositions foruse in a Salmonella nucleic acid amplification assay. In certainembodiments of the aspects provided herein, the compositions include aT7 provider oligonucleotide and a primer oligonucleotide; in which theT7 provider oligonucleotide targets a sequence in a region of Salmonellanucleic acid corresponding to bases from about 268-320 of E. coli 23SrRNA and the primer oligonucleotide targets a sequence in a region ofSalmonella nucleic acid, in which the T7 and primer oligonucleotidesused in the amplification assay target opposite strands of theSalmonella nucleic acid sequence to be amplified.

In a second aspect, there are provided compositions for use in aSalmonella nucleic acid amplification assay. In certain embodiments ofthe aspects provided herein, the compositions include a T7 provideroligonucleotide and a primer oligonucleotide; in which the T7 provideroligonucleotide targets a sequence in a region of Salmonella nucleicacid and the primer oligonucleotide targets a sequence in a region ofSalmonella nucleic acid corresponding to bases from about 338-395 of E.coli 23S rRNA, in which the T7 and primer oligonucleotides used in theamplification assay target opposite strands of the Salmonella nucleicacid sequence to be amplified.

In a third aspect, there are provided kits that include the compositionsprovided herein. In certain embodiments of the aspects provided herein,the kits include a T7 provider oligonucleotide and a primeroligonucleotide, in which the T7 provider oligonucleotide targets asequence in a region of Salmonella nucleic acid corresponding to basesfrom about 268-320 of E. coli 23S rRNA and the primer oligonucleotidetargets a sequence in a region of Salmonella nucleic acid, in which theT7 and primer oligonucleotides used in the amplification assay targetopposite strands of the Salmonella nucleic acid sequence to beamplified.

In a fourth aspect, there are provided kits that include thecompositions provided herein. In certain embodiments of the aspectsprovided herein, the kits include a T7 provider oligonucleotide and aprimer oligonucleotide, in which the T7 provider oligonucleotide targetsa sequence in a region of Salmonella nucleic acid and the primeroligonucleotide targets a sequence in a region of Salmonella nucleicacid corresponding to bases from about 338-395 of E. coli 23S rRNA, inwhich the T7 and primer oligonucleotides used in the amplification assaytarget opposite strands of the Salmonella nucleic acid sequence to beamplified.

In a fifth aspect, there are provided methods for detecting the presenceof Salmonella in a sample using the compositions and/or kits providedherein. In certain embodiments of the aspects provided herein, themethods use a T7 provider oligonucleotide and a primer oligonucleotide,in which the T7 provider oligonucleotide targets a sequence in a regionof Salmonella nucleic acid corresponding to bases from about 268-320 ofE. coli 23S rRNA and the primer oligonucleotide targets a sequence in aregion of Salmonella nucleic acid, in which the T7 and primeroligonucleotides used in the amplification assay target opposite strandsof the Salmonella nucleic acid sequence to be amplified.

In a sixth aspect, there are provided methods for detecting the presenceof Salmonella in a sample using the compositions and/or kits providedherein. In certain embodiments of the aspects provided herein, themethods use a T7 provider oligonucleotide and a primer oligonucleotide,in which the T7 provider oligonucleotide targets a sequence in a regionof Salmonella nucleic acid and the primer oligonucleotide targets asequence in a region of Salmonella nucleic acid corresponding to basesfrom about 338-395 of E. coli 23S rRNA, in which the T7 and primeroligonucleotides used in the amplification assay target opposite strandsof the Salmonella nucleic acid sequence to be amplified.

In one embodiment of the aspects provided herein, the T7 providertargets a sequence in a region of Salmonella nucleic acid correspondingto bases 268-302 of E. coli 23S rRNA. In another embodiment, the T7provider targets a sequence in a region of Salmonella nucleic acidcorresponding to bases 279-310 of E. coli 23S rRNA. In yet anotherembodiment, the T7 provider targets a sequence in a region of Salmonellanucleic acid corresponding to bases 279-309 of E. coli 23S rRNA. In aparticular embodiment, the T7 provider targets a sequence in a region ofSalmonella nucleic acid corresponding to bases 279-306 of E. coli 23SrRNA. In a certain embodiment, the T7 provider targets a sequence in aregion of Salmonella nucleic acid corresponding to bases 279-302 of E.coli 23S rRNA.

In one embodiment of the aspects provided herein, the primeroligonucleotide targets a sequence in a region of Salmonella nucleicacid corresponding to bases 349-374 of E. coli 23S rRNA. In anotherembodiment, the primer oligonucleotide targets a sequence in a region ofSalmonella nucleic acid corresponding to bases 349-370 of E. coli 23SrRNA. In yet another embodiment, the primer oligonucleotide targets asequence in a region of Salmonella nucleic acid corresponding to bases349-366 of E. coli 23S rRNA.

In certain embodiments of the aspects provided herein, the T7 provideris selected from the sequences of SEQ ID NOs: 1-34 and complements. Inother embodiments, the primer oligonucleotide is selected from thesequences of SEQ ID NOs: 35-58 and complements, as defined herein. Insome preferred embodiment, the T7 provider is selected from thesequences of SEQ ID NOs: 1-26 and complements. In other preferredembodiments, the primer oligonucleotide selected from the sequences ofSEQ ID NOs: 35-51 and complements, as defined herein.

In one particularly preferred embodiment of the aspects provided herein,the T7 provider has the sequence of SEQ ID NO: 17 or complement. Inanother particularly preferred embodiment, the primer oligonucleotidehas the sequence of SEQ ID NO: 50 or complement. In a particularlypreferred embodiment, the T7 provider has the sequence of SEQ ID NO: 26or complement. In another particularly preferred embodiment, the primeroligonucleotide has the sequence of SEQ ID NO: 49 or complement.

In certain embodiments of the compositions, methods and kits providedherein, the T7 provider oligonucleotide includes 15-35 nucleotides thatare at least 70%; or 75%; or 80%; or 85%; or 90%; or 100% complementaryto the targeted Salmonella nucleic acid sequence. In certain preferredembodiments, the T7 provider oligonucleotide includes 15-35 nucleotidesthat are complementary to the targeted Salmonella nucleic acid sequencebut have 1 mismatch; or 2 mismatches; or 3 mismatches; or 5 mismatchesas compared the targeted nucleic acid sequence within the 15-35complimentary nucleotides.

In one embodiment of the aspects provided herein, the primeroligonucleotide includes 15-35 nucleotides that are at least 70%; or75%; or 80%; or 85%; or 90% complementary to the targeted Salmonellanucleic acid sequence. In another embodiment, the primer oligonucleotideis 100% complementary to the targeted Salmonella nucleic acid sequence.In one preferred embodiment, the primer oligonucleotide includes 15-35nucleotides that are complementary to the targeted Salmonella nucleicacid sequence but have 1 mismatch; or 2 mismatches; or 3 mismatches; or5 mismatches as compared the targeted nucleic acid sequence within the15-35 complimentary nucleotides.

In some embodiments of the aspects provided herein, one or moreadditional oligonucleotide types and/or other amplification reagentsthat serve to facilitate or improve one or more aspects of thetranscription-mediated amplification reaction may be included. Forexample, in a preferred embodiment, in addition to a T7 provider and/ora primer oligonucleotide, additional oligonucleotides may furtherinclude one or more of a: detection oligonucleotide, blockeroligonucleotide, target capture oligonucleotide, and the like.

In one embodiment of the aspects provided herein, the compositions,kits, and/or methods may further include or use a detectionoligonucleotide, preferably a torch oligonucleotide or molecular beacon.In a particular embodiment, the detection oligonucleotide is a torcholigonucleotide selected from the sequences of SEQ ID NOs: 66-70 andcomplements, as defined herein. In certain preferred embodiments, thedetection oligonucleotide is a torch oligonucleotide selected from thesequences of SEQ ID NO: 66, SEQ ID NO: 67, and complements, as definedherein. In a particularly preferred embodiment, the detectionoligonucleotide is a torch oligonucleotide having the sequence of SEQ IDNO: 66 or complement, as defined herein.

In one embodiment of the aspects provided herein, the compositions,kits, and/or methods may further include or use a blockeroligonucleotide. In a particular embodiment, the blocker oligonucleotideis selected from the sequences of SEQ ID NOs: 59-65 and complements, asdefined herein. In certain preferred embodiments, the blockeroligonucleotide is selected from the sequences of SEQ ID NOs: 59, 61,63, 64, and complements, as defined herein. In a particularly preferredembodiment, the blocker oligonucleotide has the sequence of SEQ ID NOs:59 or complement, as defined herein.

In some embodiments of the aspects provided herein, the compositions,kits, and/or methods may further include or use a target captureoligonucleotide. In a particular embodiment, the target captureoligonucleotide is selected from the sequences of SEQ ID NOs: 71-77 andcomplements, as defined herein. In a preferred embodiment, the targetcapture oligonucleotide is selected from the sequences of SEQ ID NOs:71, 74, and complements as defined herein. In a particularly preferredembodiment, the target capture oligonucleotide has the sequence of SEQID NOs: 74 or complement as defined herein.

In some aspects, there are provided compositions for use in a Salmonellatranscription-mediated amplification assay (hereinafter “TMA”). In someaspects, there are provided kits for performing a Salmonellatranscription-mediated amplification assay. In some aspects, there areprovided methods for performing a Salmonella transcription-mediatedamplification assay. In certain embodiments, the compositions, kits,and/or methods may include or use one or more oligonucleotides such asa: T7 provider, primer oligonucleotide, detection oligonucleotide,blocker oligonucleotide, Torch oligonucleotide, and the like.

The terms and concepts of the invention have meanings as set forthherein unless expressly stated to the contrary and/or unless contextspecifically dictates otherwise. Unless defined otherwise, scientificand technical terms used herein have the same meaning as commonlyunderstood by those skilled in the relevant art. General definitions maybe found in technical books relevant to the art of molecular biology,e.g., Dictionary of Microbiology and Molecular Biology, 2nd ed.(Singleton et al., 1994, John Wiley & Sons, New York, N.Y.) or TheHarper Collins Dictionary of Biology (Hale & Marham, 1991, HarperPerennial, New York, N.Y.). Unless mentioned otherwise, techniquesemployed or contemplated herein are standard methodologies well known toone of ordinary skill in the art. The examples included hereinillustrate some preferred embodiments. Each reference cited herein isspecifically incorporated herein by reference in its entirety.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a nucleic acid,” is understood torepresent one or more nucleic acids. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein.

The term “nucleic acid” as used herein encompasses a singular “nucleicacid” as well as plural “nucleic acids,” and refers to any chain of twoor more nucleotides, nucleosides, or nucleobases (e.g.,deoxyribonucleotides or ribonucleotides) covalently bonded together.Nucleic acids include, but are not limited to, virus genomes, orportions thereof, either DNA or RNA, bacterial genomes, or portionsthereof, fungal, plant or animal genomes, or portions thereof, messengerRNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), plasmid DNA,mitochondrial DNA, or synthetic DNA or RNA. A nucleic acid may beprovided in a linear (e.g., mRNA), circular (e.g., plasmid), or branchedform, as well as a double-stranded or single-stranded form. Nucleicacids may include modified bases to alter the function or behavior ofthe nucleic acid, e.g., addition of a 3′-terminal dideoxynucleotide toblock additional nucleotides from being added to the nucleic acid. Asused herein, a “sequence” of a nucleic acid refers to the sequence ofbases which make up a nucleic acid.

The term “polynucleotide” as used herein denotes a nucleic acid chain.Throughout this application, nucleic acids are designated by the5′-terminus to the 3′-terminus. Standard nucleic acids, e.g., DNA andRNA, are typically synthesized “3′-to-5′,” i.e., by the addition ofnucleotides to the 5′-terminus of a growing nucleic acid.

A “nucleotide” as used herein is a subunit of a nucleic acid consistingof a phosphate group, a 5-carbon sugar and a nitrogenous base. The5-carbon sugar found in RNA is ribose. In DNA, the 5-carbon sugar is2′-deoxyribose. The term also includes analogs of such subunits, such asa methoxy group at the 2′ position of the ribose (2′-O-Me). As usedherein, methoxy oligonucleotides containing “T” residues have a methoxygroup at the 2′ position of the ribose moiety, and a uracil at the baseposition of the nucleotide.

A “non-nucleotide unit” as used herein is a unit which does notsignificantly participate in hybridization of a polymer. Such units mustnot, for example, participate in any significant hydrogen bonding with anucleotide, and would exclude units having as a component one of thefive nucleotide bases or analogs thereof.

A “target nucleic acid” as used herein is a nucleic acid comprising a“target sequence” to be amplified. Target nucleic acids may be DNA orRNA as described herein, and may be either single-stranded ordouble-stranded. The target nucleic acid may include other sequencesbesides the target sequence which may not be amplified. Typical targetnucleic acids include virus genomes, bacterial genomes, fungal genomes,plant genomes, animal genomes, rRNA, tRNA, or mRNA from viruses,bacteria or eukaryotic cells, mitochondrial DNA, or chromosomal DNA.

By “isolated” it is meant that a sample containing a target nucleic acidis taken from its natural milieu, but the term does not connote anydegree of purification.

The term “target sequence” as used herein refers to the particularnucleotide sequence of the target nucleic acid which is to be amplified.The “target sequence” includes the complexing sequences to whicholigonucleotides (e.g., priming oligonucleotides and/or promoteroligonucleotides) complex during the processes of TMA. Where the targetnucleic acid is originally single-stranded, the term “target sequence”will also refer to the sequence complementary to the “target sequence”as present in the target nucleic acid. Where the “target nucleic acid”is originally double-stranded, the term “target sequence” refers to boththe sense (+) and antisense (−) strands. In choosing a target sequence,the skilled artisan will understand that a “unique” sequence should bechosen so as to distinguish between unrelated or closely related targetnucleic acids.

The term “targets a sequence” as used herein in reference to a region ofSalmonella nucleic acid refers to a process whereby an oligonucleotidehybridizes to the target sequence in a manner that allows foramplification and detection as described herein. In one preferredembodiment, the oligonucleotide is complementary with the targetedSalmonella nucleic acid sequence and contains no mismatches. In anotherpreferred embodiment, the oligonucleotide is complementary but contains1; or 2; or 3; or 4; or 5 mismatches with the targeted Salmonellanucleic acid sequence. Preferably, the oligonucleotide that hybridizesto the Salmonella nucleic acid sequence includes at least 10 to 50; or12 to 45; or 14 to 40; or 15-35 nucleotides complementary to the targetsequence.

The term “fragment” or “region” as used herein in reference to theSalmonella targeted nucleic acid sequence refers to a piece ofcontiguous nucleic acid. In certain embodiments, the fragment includes25; or 50; or 75; or 100; or 125; or 150; or 175; or 200; or 225; or250; or 300; or 350; or 400; or 450; or 500; or 750; or 1000; or 2000;or 3000 nucleotides.

As used herein, the term “oligonucleotide” or “oligo” or “oligomer” isintended to encompass a singular “oligonucleotide” as well as plural“oligonucleotides,” and refers to any polymer of two or more ofnucleotides, nucleosides, nucleobases or related compounds used as areagent in the amplification methods disclosed herein, as well assubsequent detection methods. The oligonucleotide may be DNA and/or RNAand/or analogs thereof. The term oligonucleotide does not denote anyparticular function to the reagent, rather, it is used generically tocover all such reagents described herein. An oligonucleotide may servevarious different functions, e.g., it may function as a primer if it isspecific for and capable of hybridizing to a complementary strand andcan further be extended in the presence of a nucleic acid polymerase, itmay provide a promoter if it contains a sequence recognized by an RNApolymerase and allows for transcription (e.g., a T7 Provider), and itmay function to prevent hybridization or impede primer extension ifappropriately situated and/or modified.

As used herein, an oligonucleotide having a nucleic acid sequence“comprising” or “consisting of” or “consisting essentially of” asequence selected from a group of specific sequences means that theoligonucleotide, as a basic and novel characteristic, is capable ofstably hybridizing to a nucleic acid having the exact complement of oneof the listed nucleic acid sequences of the group under stringenthybridization conditions. An exact complement includes the correspondingDNA or RNA sequence.

As used herein, an oligonucleotide “substantially corresponding to” aspecified nucleic acid sequence means that the referred tooligonucleotide is sufficiently similar to the reference nucleic acidsequence such that the oligonucleotide has similar hybridizationproperties to the reference nucleic acid sequence in that it wouldhybridize with the same target nucleic acid sequence under stringenthybridization conditions. One skilled in the art will understand that“substantially corresponding oligonucleotides” can vary from thereferred to sequence and still hybridize to the same target nucleic acidsequence. This variation from the nucleic acid may be stated in terms ofa percentage of identical bases within the sequence or the percentage ofperfectly complementary bases between the probe or primer and its targetsequence. Thus, an oligonucleotide “substantially corresponds” to areference nucleic acid sequence if these percentages of base identity orcomplementarity are from 100% to about 80%. In preferred embodiments,the percentage is from 100% to about 85%. In more preferred embodiments,this percentage can be from 100% to about 90%; in other preferredembodiments, this percentage is from 100% to about 95%. One skilled inthe art will understand the various modifications to the hybridizationconditions that might be required at various percentages ofcomplementarity to allow hybridization to a specific target sequencewithout causing an unacceptable level of non-specific hybridization.

A “helper oligonucleotide” or “helper” refers to an oligonucleotidedesigned to bind to a target nucleic acid and impose a differentsecondary and/or tertiary structure on the target to increase the rateand extent of hybridization of a detection probe or otheroligonucleotide with the targeted nucleic acid, as described, forexample, in U.S. Pat. No. 5,030,557, the contents of which areincorporated by reference herein. Helpers may also be used to assistwith the hybridization to target nucleic acid sequences and function ofprimer, target capture and other oligonucleotides.

As used herein, a “blocking moiety” is a substance used to “block” the3′-terminus of an oligonucleotide or other nucleic acid so that itcannot be efficiently extended by a nucleic acid polymerase.

As used herein, a “priming oligonucleotide” or “primer” is anoligonucleotide, at least the 3′-end of which is complementary to anucleic acid template, and which complexes (by hydrogen bonding orhybridization) with the template to give a primer: template complexsuitable for initiation of synthesis by an RNA- or DNA-dependent DNApolymerase.

As used herein, a “promoter” is a specific nucleic acid sequence that isrecognized by a DNA-dependent RNA polymerase (“transcriptase”) as asignal to bind to the nucleic acid and begin the transcription of RNA ata specific site.

As used herein, a “promoter-provider” or “provider” refers to anoligonucleotide comprising first and second regions, and which ismodified to prevent the initiation of DNA synthesis from its3′-terminus. The “first region” of a promoter-provider oligonucleotidecomprises a base sequence which hybridizes to a DNA template, where thehybridizing sequence is situated 3′, but not necessarily adjacent to, apromoter region. The hybridizing portion of a promoter oligonucleotideis typically at least 10 nucleotides in length, and may extend up to 15,20, 25, 30, 35, 40, 50 or more nucleotides in length. The “secondregion” comprises a promoter sequence for an RNA polymerase. Apromoter-provider oligonucleotide is engineered so that it is incapableof being extended by an RNA -or DNA-dependent DNA polymerase, e.g.,reverse transcriptase, preferably comprising a blocking moiety at its3′-terminus as described above. As referred to herein, a “T7 provider”is a blocked promoter-provider oligonucleotide that provides anoligonucleotide sequence that is recognized by T7 RNA polymerase.

As used herein, a “terminating oligonucleotide” or “blockeroligonucleotide” is an oligonucleotide comprising a base sequence thatis complementary to a region of the target nucleic acid in the vicinityof the 5′-end of the target sequence, so as to “terminate” primerextension of a nascent nucleic acid that includes a primingoligonucleotide, thereby providing a defined 3′-end for the nascentnucleic acid strand.

An “extender oligonucleotide” or “extend oligo” as used herein refers toan oligonucleotide that is the same sense as the T7 Provider and may actas a helper oligonucleotide that opens up structure or improvesspecificity.

As used herein, a “detection oligonucleotide” refers to a nucleic acidoligonucleotide that hybridizes specifically to a target sequence,including an amplified sequence, under conditions that promote nucleicacid hybridization, for detection of the target nucleic acid. By “probe”or “detection probe” is meant a molecule comprising an oligonucleotidehaving a base sequence partly or completely complementary to a region ofa target sequence sought to be detected, so as to hybridize theretounder stringent hybridization conditions.

By “stable” or “stable for detection” is meant that the temperature of areaction mixture is at least 2° C. below the melting temperature of anucleic acid duplex.

By “amplification” or “nucleic acid amplification” is meant productionof multiple copies of a target nucleic acid that contains at least aportion of the intended specific target nucleic acid sequence, asfurther described herein. The multiple copies may be referred to asamplicons or amplification products.

The term “amplicon” as used herein refers to the nucleic acid moleculegenerated during an amplification procedure that is complementary orhomologous to a sequence contained within the target sequence.

By “preferentially hybridize” is meant that under stringenthybridization assay conditions, probes hybridize to their targetsequences, or replicates thereof, to form stable probe: target hybrids,while at the same time formation of stable probe: non-target hybrids isminimized. Thus, a probe hybridizes to a target sequence or replicatethereof to a sufficiently greater extent than to a non-target sequence,to enable one having ordinary skill in the art to accurately quantitatethe RNA replicates or complementary DNA (cDNA) of the target sequenceformed during the amplification.

By “complementary” is meant that the nucleotide sequences of similarregions of two single-stranded nucleic acids, or to different regions ofthe same single-stranded nucleic acid have a nucleotide base compositionthat allow the single-stranded regions to hybridize together in a stabledouble-stranded hydrogen-bonded region under stringent hybridization oramplification conditions. When a contiguous sequence of nucleotides ofone single-stranded region is able to form a series of “canonical”hydrogen-bonded base pairs with an analogous sequence of nucleotides ofthe other single-stranded region, such that A is paired with U or T andC is paired with G, the nucleotides sequences are “perfectly”complementary.

By “nucleic acid hybrid” or “hybrid” or “duplex” is meant a nucleic acidstructure containing a double-stranded, hydrogen-bonded region whereineach strand is complementary to the other, and wherein the region issufficiently stable under stringent hybridization conditions to bedetected by means including, but not limited to, chemiluminescent orfluorescent light detection, autoradiography, or gel electrophoresis.Such hybrids may comprise RNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

As used herein, a “capture oligonucleotide” or “capture probe” refers toa nucleic acid oligomer that specifically hybridizes to a targetsequence in a target nucleic acid by standard base pairing and joins toa binding partner on an immobilized probe to capture the target nucleicacid to a support. One example of a capture oligomer includes twobinding regions: a sequence-binding region (i.e., target-specificportion) and an immobilized probe-binding region, usually on the sameoligomer, although the two regions may be present on two differentoligomers joined together by one or more linkers.

As used herein, an “immobilized oligonucleotide”, “immobilized probe” or“immobilized nucleic acid” refers to a nucleic acid binding partner thatjoins a capture oligomer to a support, directly or indirectly. Animmobilized probe joined to a support facilitates separation of acapture probe bound target from unbound material in a sample.

As used herein, a “label” refers to a moiety or compound joined directlyor indirectly to a probe that is detected or leads to a detectablesignal.

As used herein, structures referred to as “molecular torches” aredesigned to include distinct regions of self-complementarity (coined“the target binding domain” and “the target closing domain”) which areconnected by a joining region and which hybridize to one another underpredetermined hybridization assay conditions.

As used herein, a “DNA-dependent DNA polymerase” is an enzyme thatsynthesizes a complementary DNA copy from a DNA template. Examples areDNA polymerase I from E. coli, bacteriophage T7 DNA polymerase, or DNApolymerases from bacteriophages T4, Phi-29, M2, or T5. DNA-dependent DNApolymerases may be the naturally occurring enzymes isolated frombacteria or bacteriophages or expressed recombinantly, or may bemodified or “evolved” forms which have been engineered to possesscertain desirable characteristics, e.g., thermostability, or the abilityto recognize or synthesize a DNA strand from various modified templates.All known DNA-dependent DNA polymerases require a complementary primerto initiate synthesis. It is known that under suitable conditions aDNA-dependent DNA polymerase may synthesize a complementary DNA copyfrom an RNA template. RNA-dependent DNA polymerases typically also haveDNA-dependent DNA polymerase activity.

As used herein, a “DNA-dependent RNA polymerase” or “transcriptase” isan enzyme that synthesizes multiple RNA copies from a double-stranded orpartially-double-stranded DNA molecule having a promoter sequence thatis usually double-stranded. The RNA molecules (“transcripts”) aresynthesized in the 5′-to-3′ direction beginning at a specific positionjust downstream of the promoter. Examples of transcriptases are the DNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, andSP6.

As used herein, an “RNA-dependent DNA polymerase” or “reversetranscriptase” (“RT”) is an enzyme that synthesizes a complementary DNAcopy from an RNA template. All known reverse transcriptases also havethe ability to make a complementary DNA copy from a DNA template; thus,they are both RNA- and DNA-dependent DNA polymerases. RTs may also havean RNAse H activity. A primer is required to initiate synthesis withboth RNA and DNA templates.

As used herein, a “selective RNAse” is an enzyme that degrades the RNAportion of an RNA:DNA duplex but not single-stranded RNA,double-stranded RNA or DNA. An exemplary selective RNAse is RNAse H.Enzymes other than RNAse H which possess the same or similar activitymay also be used. Selective RNAses may be endonucleases or exonucleases.Most reverse transcriptase enzymes contain an RNAse H activity inaddition to their polymerase activities. However, other sources of theRNAse H are available without an associated polymerase activity. Thedegradation may result in separation of RNA from a RNA:DNA complex.Alternatively, a selective RNAse may simply cut the RNA at variouslocations such that portions of the RNA melt off or permit enzymes tounwind portions of the RNA. Other enzymes which selectively degrade RNAtarget sequences or RNA products of the present invention will bereadily apparent to those of ordinary skill in the art.

The term “specificity,” in the context of an amplification system, isused herein to refer to the characteristic of an amplification systemwhich describes its ability to distinguish between target and non-targetsequences dependent on sequence and assay conditions. In terms of anucleic acid amplification, specificity generally refers to the ratio ofthe number of specific amplicons produced to the number of side-products(i.e., the signal-to-noise ratio).

The term “sensitivity” is used herein to refer to the precision withwhich a nucleic acid amplification reaction can be detected orquantitated. The sensitivity of an amplification reaction is generally ameasure of the smallest copy number of the target nucleic acid that canbe reliably detected in the amplification system, and will depend, forexample, on the detection assay being employed, and the specificity ofthe amplification reaction, i.e., the ratio of specific amplicons toside-products.

As used herein, a “colony forming unit” (“CFU”) is used as a measure ofviable microorganisms in a sample. A CFU is an individual viable cellcapable of forming on a solid medium a visible colony whose individualcells are derived by cell division from one parental cell. One CFUcorresponds to ˜1000 copies of rRNA.

As used herein, the term “TTime” is the threshold time or time ofemergence of signal in a real-time plot of the assay data. TTime valuesestimate the time at which a particular threshold indicating ampliconproduction is passed in a real-time amplification reaction. TTime and analgorithm for calculating and using TTime values are described in Lightet al., U.S. Pub. No. 2006/0276972, paragraphs [0517] through [0538],the disclosure of which is hereby incorporated by reference herein. Acurve fitting procedure is applied to normalized and background-adjusteddata. The curve fit is performed for only a portion of the data betweena predetermined low bound and high bound. The goal, after finding thecurve that fits the data, is to estimate the time corresponding to thepoint at which the curve or a projection thereof intersects a predefinedthreshold value. In one embodiment, the threshold for normalized data is0.11. The high and low bounds are determined empirically as that rangeover which curves fit to a variety of control data sets exhibit theleast variability in the time associated with the given threshold value.For example, in one embodiment, the low bound is 0.04 and the high boundis 0.36. The curve is fit for data extending from the first data pointbelow the low bound through the first data point past the high bound.Next, there is made a determination whether the slope of the fit isstatistically significant. For example, if the p value of the firstorder coefficient is less than 0.05, the fit is considered significant,and processing continues. If not, processing stops. Alternatively, thevalidity of the data can be determined by the R² value. The slope m andintercept b of the linear curve y=mx+b are determined for the fittedcurve. With that information, TTime can be determined as follows:TTime=(Threshold−b)/m

As used herein, the term “relative fluorescence unit” (“RFU”) is anarbitrary unit of measurement of fluorescence intensity. RFU varies withthe characteristics of the detection means used for the measurement.

As used herein, the term “real-time TMA” refers to single-primertranscription-mediated amplification (“TMA”) of target nucleic acid thatis monitored by real-time detection means.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B illustrate real-time amplification charts of analyte showing(FIG. 1A) “Poor” and (FIG. 1B) “Good” assay performance of differentcombinations of amplification and detection oligonucleotides. Theanalyte used was purified Salmonella enterica rRNA and the charts showmultiple replicates of the analyte at 0, 1E+4, and 1E+5 copies.

FIG. 2 shows Salmonella enterica sbsp enterica sv Enteritidis GP60(ATCC13076) “350 region” sequence (SEQ ID NO: 150) corresponding tonucleotides 150-425 of E. coli 23s rRNA sequence.

DETAILED DESCRIPTION OF THE INVENTION

In certain aspects and embodiments, the invention relates tocompositions, methods and kits for the identification, detection, and/orquantitation of Salmonella, which may be present either alone or as acomponent, large or small, of a homogeneous or heterogeneous mixture ofnucleic acids in a sample taken for testing, e.g., for diagnostictesting, for screening of blood products, for microbiological detectionin bioprocesses, food, water, industrial or environmental samples, andfor other purposes. Specific methods, compositions, and kits asdisclosed herein provide improved sensitivity, specificity, or speed ofdetection in the amplification-based detection of Salmonella. Salmonellaribosomal RNA is very closely related to E. coli, Shigella sp.,Citrobacter sp., Enterobacter sp and other potential enteric bacteria.Accordingly, in certain embodiments of the invention, the Salmonellaassay identifies rRNA sequences common to nearly all species, subspeciesand serovars of the Salmonella genus, and differentiates Salmonella fromother enteric bacteria. A useful region for such differentiation is the350 region of the 23S rRNA.

As a result of extensive analyses of amplification oligonucleotidesspecific for Salmonella, the particular region of Salmonella,corresponding to the region of E. coli 23s rRNA reference sequence(accession no. AJ278710) from about 150-425 nucleotide bases(hereinafter referred to as the “350 region”), has been identified as apreferred target for amplification-based detection of Salmonella.Accordingly, the invention relates to methods of detection of Salmonellain a sample of interest, amplification oligonucleotides, compositions,reactions mixtures, kits, and the like.

The Salmonella genus assay detects ribosomal RNA sequences specific forknown Salmonella species. It utilizes real-time TMA technology, wherethe target-specific sequence is amplified using reverse TMA and afluorescent molecular torch is used to detect the amplified products asthey are produced. Target detection is performed simultaneously with theamplification and detection of an internal control in order to confirmreliability of the result. The result of the assay consists of theclassification of the sample as positive or negative for the presence orabsence of Salmonella.

In one embodiment, the sample is a biopharmaceutical process(bioprocess) stream where Salmonella is a known or suspectedcontaminant. A “bioprocess,” as used herein, refers generally to anyprocess in which living cells or organisms, or components thereof, arepresent, either intended or unintended. For example, essentially anymanufacturing or other process that employs one or more samples orsample streams, at least one of which contains living cells, organisms,or components thereof, or contains such cells, organisms or componentsas a result of unintended contamination, is considered a bioprocess. Inmany such processes it is desirable to have the ability to detect,identify and/or control the presence and/or sources of living cells,organisms or components thereof within a process. Using the methodsdisclosed herein, for example, the presence and/or sources of Salmonellain one or more bioprocess samples and/or streams may be monitored in arapid and sensitive fashion.

Target Nucleic Acid/Target Sequence

Target nucleic acids may be isolated from any number of sources based onthe purpose of the amplification assay being carried out. Sources oftarget nucleic acids include, but are not limited to, clinicalspecimens, e.g., blood, urine, saliva, feces, semen, or spinal fluid,from criminal evidence, from environmental samples, e.g., water or soilsamples, from food, from industrial samples, from cDNA libraries, orfrom total cellular RNA. If necessary, target nucleic acids are madeavailable for interaction with various oligonucleotides. This mayinclude, for example, cell lysis or cell permeabilization to release thetarget nucleic acid from cells, which then may be followed by one ormore purification steps, such as a series of isolation and wash steps.See, e.g., Clark et al., “Method for Extracting Nucleic Acids from aWide Range of Organisms,” U.S. Pat. No. 5,786,208, the contents of whichare hereby incorporated by reference herein. This is particularlyimportant where the sample may contain components that can interferewith the amplification reaction, such as, for example, heme present in ablood sample. See Ryder et al., “Amplification of Nucleic Acids fromMononuclear Cells Using Iron Complexing and Other Agents,” U.S. Pat. No.5,639,599, the contents of which are hereby incorporated by referenceherein. Methods to prepare target nucleic acids from various sources foramplification are well known to those of ordinary skill in the art.Target nucleic acids may be purified to some degree prior to theamplification reactions described herein, but in other cases, the sampleis added to the amplification reaction without any furthermanipulations.

As will be understood by those of ordinary skill in the art, “unique”sequences are judged from the testing environment. At least thesequences recognized by the detection probe should be unique in theenvironment being tested, but need not be unique within the universe ofall possible sequences. Furthermore, even though the target sequenceshould contain a “unique” sequence for recognition by a detection probe,it is not always the case that the priming oligonucleotide and/orpromoter oligonucleotide are recognizing “unique” sequences. In someembodiments, it may be desirable to choose a target sequence which iscommon to a family of related organisms. In other situations, a veryhighly specific target sequence, or a target sequence having at least ahighly specific region recognized by the detection probe andamplification oligonucleotides, would be chosen so as to distinguishbetween closely related organisms, for example, between pathogenic andnon-pathogenic E. coli. A target sequence may be of any practicallength. A minimal target sequence includes the region which hybridizesto the priming oligonucleotide (or the complement thereof), the regionwhich hybridizes to the hybridizing region of the promoteroligonucleotide (or the complement thereof), and a region used fordetection, e.g., a region which hybridizes to a detection probe. Theregion which hybridizes with the detection probe may overlap with or becontained within the region which hybridizes with the primingoligonucleotide (or its complement) or the hybridizing region of thepromoter oligonucleotide (or its complement). In addition to the minimalrequirements, the optimal length of a target sequence depends on anumber of considerations, for example, the amount of secondarystructure, or self-hybridizing regions in the sequence. Typically,target sequences range from about 30 nucleotides in length to about 300nucleotides in length. The optimal or preferred length may vary underdifferent conditions which can be determined according to the methodsdescribed herein.

Nucleic Acid “Identity”

In certain embodiments, a nucleic acid comprises a contiguous baseregion that is at least 70%; or 75%; or 80%, or 85% or 90%, or 95%; or100% identical to a contiguous base region of a reference nucleic acid.For short nucleic acids, the degree of identity between a base region ofa “query” nucleic acid and a base region of a reference nucleic acid canbe determined by manual alignment. “Identity” is determined by comparingjust the sequence of nitrogenous bases, irrespective of the sugar andbackbone regions of the nucleic acids being compared. Thus, thequery:reference base sequence alignment may be DNA:DNA, RNA:RNA,DNA:RNA, RNA:DNA, or any combinations or analogs thereof. Equivalent RNAand DNA base sequences can be compared by converting U's (in RNA) to T's(in DNA).

Oligonucleotides & Primers

An oligonucleotide can be virtually any length, limited only by itsspecific function in the amplification reaction or in detecting anamplification product of the amplification reaction. However, in certainembodiments, preferred oligonucleotides will contain at least about 10;or 12; or 14; or 16; or 18; or 20; or 22; or 24; or 26; or 28; or 30; or32; or 34; or 36; or 38; or 40; or 42; or 44; or 46; or 48; or 50; or52; or 54; or 56 contiguous bases that are complementary to a region ofthe target nucleic acid sequence or its complementary strand. Thecontiguous bases are preferably at least about 80%, more preferably atleast about 90%, and most preferably completely complementary to thetarget sequence to which the oligonucleotide binds. Certain preferredoligonucleotides are of lengths generally between about 10-100; or12-75; or 14-50; or 15-40 bases long and optionally can include modifiednucleotides.

Oligonucleotides of a defined sequence and chemical structure may beproduced by techniques known to those of ordinary skill in the art, suchas by chemical or biochemical synthesis, and by in vitro or in vivoexpression from recombinant nucleic acid molecules, e.g., bacterial orviral vectors. As intended by this disclosure, an oligonucleotide doesnot consist solely of wild-type chromosomal DNA or the in vivotranscription products thereof.

Oligonucleotides may be modified in any way, as long as a givenmodification is compatible with the desired function of a givenoligonucleotide. One of ordinary skill in the art can easily determinewhether a given modification is suitable or desired for any givenoligonucleotide. Modifications include base modifications, sugarmodifications or backbone modifications. Base modifications include, butare not limited to the use of the following bases in addition toadenine, cytidine, guanosine, thymine and uracil: C-5 propyne, 2-aminoadenine, 5-methyl cytidine, inosine, and dP and dK bases. The sugargroups of the nucleoside subunits may be ribose, deoxyribose and analogsthereof, including, for example, ribonucleosides having a 2′-O-methylsubstitution to the ribofuranosyl moiety. See Becker et al., U.S. Pat.No. 6,130,038. Other sugar modifications include, but are not limited to2′-amino, 2′-fluoro, (L)-alpha-threofuranosyl, and pentopyranosylmodifications. The nucleoside subunits may by joined by linkages such asphosphodiester linkages, modified linkages or by non-nucleotide moietieswhich do not prevent hybridization of the oligonucleotide to itscomplementary target nucleic acid sequence. Modified linkages includethose linkages in which a standard phosphodiester linkage is replacedwith a different linkage, such as a phosphorothioate linkage or amethylphosphonate linkage. The nucleobase subunits may be joined, forexample, by replacing the natural deoxyribose phosphate backbone of DNAwith a pseudo peptide backbone, such as a 2-aminoethylglycine backbonewhich couples the nucleobase subunits by means of a carboxymethyl linkerto the central secondary amine. DNA analogs having a pseudo peptidebackbone are commonly referred to as “peptide nucleic acids” or “PNA”and are disclosed by Nielsen et al., “Peptide Nucleic Acids,” U.S. Pat.No. 5,539,082. Other linkage modifications include, but are not limitedto, morpholino bonds.

Non-limiting examples of oligonucleotides or oligos contemplated hereininclude nucleic acid analogs containing bicyclic and tricyclicnucleoside and nucleotide analogs (LNAs). See Imanishi et al., U.S. Pat.No. 6,268,490; and Wengel et al., U.S. Pat. No. 6,670,461.) Any nucleicacid analog is contemplated by the present invention provided themodified oligonucleotide can perform its intended function, e.g.,hybridize to a target nucleic acid under stringent hybridizationconditions or amplification conditions, or interact with a DNA or RNApolymerase, thereby initiating extension or transcription. In the caseof detection probes, the modified oligonucleotides must also be capableof preferentially hybridizing to the target nucleic acid under stringenthybridization conditions.

The design and sequence of oligonucleotides depend on their function asdescribed below. Several variables to take into account include: length,melting temperature (Tm), specificity, complementarity with otheroligonucleotides in the system, G/C content, polypyrimidine (T, C) orpolypurine (A, G) stretches, and the 3′-end sequence. Controlling forthese and other variables is a standard and well known aspect ofoligonucleotide design, and various computer programs are readilyavailable to initially screen large numbers of potentialoligonucleotides.

The 3′-terminus of an oligonucleotide (or other nucleic acid) can beblocked in a variety of ways using a blocking moiety, as describedbelow. A “blocked” oligonucleotide is not efficiently extended by theaddition of nucleotides to its 3′-terminus, by a DNA- or RNA-dependentDNA polymerase, to produce a complementary strand of DNA. As such, a“blocked” oligonucleotide cannot be a “primer.”

Blocking Moiety

A blocking moiety may be a small molecule, e.g., a phosphate or ammoniumgroup, or it may be a modified nucleotide, e.g., a 3′2′dideoxynucleotide or 3′ deoxyadenosine 5′-triphosphate (cordycepin), orother modified nucleotide. Additional blocking moieties include, forexample, the use of a nucleotide or a short nucleotide sequence having a3′-to-5′ orientation, so that there is no free hydroxyl group at the3′-terminus, the use of a 3′ alkyl group, a 3′ non-nucleotide moiety(see, e.g., Arnold et al., “Non-Nucleotide Linking Reagents forNucleotide Probes,” U.S. Pat. No. 6,031,091, the contents of which arehereby incorporated by reference herein), phosphorothioate, alkane-diolresidues, peptide nucleic acid (PNA), nucleotide residues lacking a 3′hydroxyl group at the 3′-terminus, or a nucleic acid binding protein.Preferably, the 3′-blocking moiety comprises a nucleotide or anucleotide sequence having a 3′-to-5′ orientation or a 3′ non-nucleotidemoiety, and not a 3′2′-dideoxynucleotide or a 3′ terminus having a freehydroxyl group. Additional methods to prepare 3′-blockingoligonucleotides are well known to those of ordinary skill in the art.

Priming Oligonucleotide or Primer

A priming oligonucleotide is extended by the addition of covalentlybonded nucleotide bases to its 3′-terminus, which bases arecomplementary to the template. The result is a primer extension product.Suitable and preferred priming oligonucleotides are described herein.Virtually all DNA polymerases (including reverse transcriptases) thatare known require complexing of an oligonucleotide to a single-strandedtemplate (“priming”) to initiate DNA synthesis, whereas RNA replicationand transcription (copying of RNA from DNA) generally do not require aprimer. By its very nature of being extended by a DNA polymerase, apriming oligonucleotide does not comprise a 3′-blocking moiety.

Promoter Oligonucleotide/Promoter Sequence

For binding, it was generally thought that such transcriptases requiredDNA which had been rendered double-stranded in the region comprising thepromoter sequence via an extension reaction, however, it has beendetermined that efficient transcription of RNA can take place even underconditions where a double-stranded promoter is not formed through anextension reaction with the template nucleic acid. The template nucleicacid (the sequence to be transcribed) need not be double-stranded.Individual DNA-dependent RNA polymerases recognize a variety ofdifferent promoter sequences, which can vary markedly in theirefficiency in promoting transcription. When an RNA polymerase binds to apromoter sequence to initiate transcription, that promoter sequence isnot part of the sequence transcribed. Thus, the RNA transcripts producedthereby will not include that sequence.

Terminating Oligonucleotide

A terminating oligonucleotide or “blocker” is designed to hybridize tothe target nucleic acid at a position sufficient to achieve the desired3′-end for the nascent nucleic acid strand. The positioning of theterminating oligonucleotide is flexible depending upon its design. Aterminating oligonucleotide may be modified or unmodified. In certainembodiments, terminating oligonucleotides are synthesized with at leastone or more 2-O-methyl ribonucleotides. These modified nucleotides havedemonstrated higher thermal stability of complementary duplexes. The2′-O-methyl ribonucleotides also function to increase the resistance ofoligonucleotides to exonucleases, thereby increasing the half-life ofthe modified oligonucleotides. See, e.g., Majlessi et al. (1988) NucleicAcids Res. 26, 2224-9, the contents of which are hereby incorporated byreference herein. Other modifications as described elsewhere herein maybe utilized in addition to or in place of 2′-O-methyl ribonucleotides.For example, a terminating oligonucleotide may comprise PNA or an LNA.See, e.g., Petersen et al. (2000) J. Mol. Recognit. 13, 44-53, thecontents of which are hereby incorporated by reference herein. Aterminating oligonucleotide typically includes a blocking moiety at its3′-terminus to prevent extension. A terminating oligonucleotide may alsocomprise a protein or peptide joined to the oligonucleotide so as toterminate further extension of a nascent nucleic acid chain by apolymerase. Suitable and preferred terminating oligonucleotides aredescribed herein. It is noted that while a terminating oligonucleotidetypically or necessarily includes a 3′-blocking moiety, “3′-blocked”oligonucleotides are not necessarily terminating oligonucleotides. Otheroligonucleotides as disclosed herein, e.g., promoter oligonucleotidesand capping oligonucleotides are typically or necessarily 3′-blocked aswell.

Extender Oligonucleotide

An extender oligonucleotide hybridizes to a DNA template adjacent to ornear the 3′-end of the first region of a promoter oligonucleotide. Anextender oligonucleotide preferably hybridizes to a DNA template suchthat the 5′-terminal base of the extender oligonucleotide is within 3, 2or 1 bases of the 3′-terminal base of a promoter oligonucleotide. Mostpreferably, the 5′-terminal base of an extender oligonucleotide isadjacent to the 3′-terminal base of a promoter oligonucleotide when theextender oligonucleotide and the promoter oligonucleotide are hybridizedto a DNA template. To prevent extension of an extender oligonucleotide,a 3′-terminal blocking moiety is typically included.

Probe

As would be understood by someone having ordinary skill in the art, aprobe comprises an isolated nucleic acid molecule, or an analog thereof,in a form not found in nature without human intervention (e.g.,recombined with foreign nucleic acid, isolated, or purified to someextent). Probes may have additional nucleosides or nucleobases outsideof the targeted region so long as such nucleosides or nucleobases do notsubstantially affect hybridization under stringent hybridizationconditions and, in the case of detection probes, do not preventpreferential hybridization to the target nucleic acid. Anon-complementary sequence may also be included, such as a targetcapture sequence (generally a homopolymer tract, such as a poly-A,poly-T or poly-U tail), promoter sequence, a binding site for RNAtranscription, a restriction endonuclease recognition site, or maycontain sequences which will confer a desired secondary or tertiarystructure, such as a catalytic active site or a hairpin structure on theprobe, on the target nucleic acid, or both.

The probes preferably include at least one detectable label. The labelmay be any suitable labeling substance, including but not limited to aradioisotope, an enzyme, an enzyme cofactor, an enzyme substrate, a dye,a hapten, a chemiluminescent molecule, a fluorescent molecule, aphosphorescent molecule, an electrochemiluminescent molecule, achromophore, a base sequence region that is unable to stably hybridizeto the target nucleic acid under the stated conditions, and mixtures ofthese. In one particularly preferred embodiment, the label is anacridinium ester. Certain probes as disclosed herein do not include alabel. For example, non-labeled “capture” probes may be used to enrichfor target sequences or replicates thereof, which may then be detectedby a second “detection” probe. See, e.g., Weisburg et al., “Two-StepHybridization and Capture of a Polynucleotide,” U.S. Pat. No. 6,534,273,which is hereby incorporated by reference herein. While detection probesare typically labeled, certain detection technologies do not requirethat the probe be labeled. See, e.g., Nygren et al., “Devices andMethods for Optical Detection of Nucleic Acid Hybridization,” U.S. Pat.No. 6,060,237.

Probes of a defined sequence may be produced by techniques known tothose of ordinary skill in the art, such as by chemical synthesis, andby in vitro or in vivo expression from recombinant nucleic acidmolecules. Preferably probes are 10 to 100 nucleotides in length, morepreferably 12 to 50 bases in length, and even more preferably 18 to 35bases in length.

Hybridize/Hybridization

Nucleic acid hybridization is the process by which two nucleic acidstrands having completely or partially complementary nucleotidesequences come together under predetermined reaction conditions to forma stable, double-stranded hybrid. Either nucleic acid strand may be adeoxyribonucleic acid (DNA) or a ribonucleic acid (RNA) or analogsthereof. Thus, hybridization can involve RNA:RNA hybrids, DNA:DNAhybrids, RNA:DNA hybrids, or analogs thereof The two constituent strandsof this double-stranded structure, sometimes called a hybrid, are heldtogether by hydrogen bonds. Although these hydrogen bonds most commonlyform between nucleotides containing the bases adenine and thymine oruracil (A and T or U) or cytosine and guanine (C and G) on singlenucleic acid strands, base pairing can also form between bases which arenot members of these “canonical” pairs. Non-canonical base pairing iswell-known in the art. (See, e.g., Roger L. P. Adams et al., “TheBiochemistry Of The Nucleic Acids” (11^(th) ed. 1992).)

“Stringent” hybridization assay conditions refer to conditions wherein aspecific detection probe is able to hybridize with target nucleic acidsover other nucleic acids present in the test sample. It will beappreciated that these conditions may vary depending upon factorsincluding the GC content and length of the probe, the hybridizationtemperature, the composition of the hybridization reagent or solution,and the degree of hybridization specificity sought. Specific stringenthybridization conditions are provided in the disclosure below.

Nucleic Acid Amplification

Many well-known methods of nucleic acid amplification requirethermocycling to alternately denature double-stranded nucleic acids andhybridize primers; however, other well-known methods of nucleic acidamplification are isothermal. The polymerase chain reaction (U.S. Pat.Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188), commonly referred toas PCR, uses multiple cycles of denaturation, annealing of primer pairsto opposite strands, and primer extension to exponentially increase copynumbers of the target sequence. In a variation called RT-PCR, reversetranscriptase (RT) is used to make a complementary DNA (cDNA) from mRNA,and the cDNA is then amplified by PCR to produce multiple copies of DNA.The ligase chain reaction (Weiss, R. 1991, Science 254: 1292), commonlyreferred to as LCR, uses two sets of complementary DNA oligonucleotidesthat hybridize to adjacent regions of the target nucleic acid. The DNAoligonucleotides are covalently linked by a DNA ligase in repeatedcycles of thermal denaturation, hybridization and ligation to produce adetectable double-stranded ligated oligonucleotide product. Anothermethod is strand displacement amplification (Walker, G. et al., 1992,Proc. Natl. Acad. Sci. USA 89:392-396; U.S. Pat. Nos. 5,270,184 and5,455,166), commonly referred to as SDA, which uses cycles of annealingpairs of primer sequences to opposite strands of a target sequence,primer extension in the presence of a dNTPaS to produce a duplexhemiphosphorothioated primer extension product, endonuclease-mediatednicking of a hemimodified restriction endonuclease recognition site, andpolymerase-mediated primer extension from the 3′ end of the nick todisplace an existing strand and produce a strand for the next round ofprimer annealing, nicking and strand displacement, resulting ingeometric amplification of product. Thermophilic SDA (tSDA) usesthermophilic endonucleases and polymerases at higher temperatures inessentially the same method (European Pat. No. 0 684 315). Otheramplification methods include: nucleic acid sequence based amplification(U.S. Pat. No. 5,130,238), commonly referred to as NASBA; one that usesan RNA replicase to amplify the probe molecule itself (Lizardi, P. etal., 1988, BioTechnol. 6: 1197-1202), commonly referred to as Q-βreplicase; a transcription-based amplification method (Kwoh, D. et al.,1989, Proc. Natl. Acad. Sci. USA 86:1173-1177); self-sustained sequencereplication (Guatelli, J. et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878); and, transcription-mediated amplification (U.S. Pat. Nos.5,480,784 and 5,399,491), commonly referred to as TMA. For furtherdiscussion of known amplification methods see Persing, David H., 1993,“In Vitro Nucleic Acid Amplification Techniques” in Diagnostic MedicalMicrobiology: Principles and Applications (Persing et al., Eds.), pp.51-87 (American Society for Microbiology, Washington, D.C.).

In a preferred embodiment, Salmonella is detected by atranscription-based amplification technique. One preferredtranscription-based amplification system is transcription-mediatedamplification (TMA), which employs an RNA polymerase to produce multipleRNA transcripts of a target region. Exemplary TMA amplification methodsare described in U.S. Pat. Nos. 5,480,784, 5,399,491, 7,374,885, andreferences cited therein, the contents of which are incorporated hereinby reference in their entireties. TMA uses a “promoter-primer” thathybridizes to a target nucleic acid in the presence of a reversetranscriptase and an RNA polymerase to form a double-stranded promoterfrom which the RNA polymerase produces RNA transcripts. Thesetranscripts can become templates for further rounds of TMA in thepresence of a second primer capable of hybridizing to the RNAtranscripts. Unlike PCR, LCR or other methods that require heatdenaturation, TMA is an isothermal method that uses an RNase H activityto digest the RNA strand of an RNA:DNA hybrid, thereby making the DNAstrand available for hybridization with a primer or promoter-primer.Generally, the RNase H activity associated with the reversetranscriptase provided for amplification is used.

In one version of the TMA method, one amplification primer is anoligonucleotide promoter-primer that comprises a promoter sequence whichbecomes functional when double-stranded, located 5′ of a target-bindingsequence, which is capable of hybridizing to a binding site of a targetRNA at a location 3′ to the sequence to be amplified. A promoter-primermay be referred to as a “T7-primer” when it is specific for T7 RNApolymerase recognition. Under certain circumstances, the 3′ end of apromoter-primer, or a subpopulation of such promoter-primers, may bemodified to block or reduce promoter-primer extension. From anunmodified promoter-primer, reverse transcriptase creates a cDNA copy ofthe target RNA, while RNase H activity degrades the target RNA. A secondamplification primer then binds to the cDNA. This primer may be referredto as a “non-T7 primer” to distinguish it from a “T7-primer”. From thissecond amplification primer, reverse transcriptase creates another DNAstrand, resulting in a double-stranded DNA with a functional promoter atone end. When double-stranded, the promoter sequence is capable ofbinding an RNA polymerase to begin transcription of the target sequenceto which the promoter-primer is hybridized. An RNA polymerase uses thispromoter sequence to produce multiple RNA transcripts (i.e., amplicons),generally about 100 to 1,000 copies. Each newly-synthesized amplicon cananneal with the second amplification primer. Reverse transcriptase canthen create a DNA copy, while the RNase H activity degrades the RNA ofthis RNA:DNA duplex. The promoter-primer can then bind to the newlysynthesized DNA, allowing the reverse transcriptase to create adouble-stranded DNA, from which the RNA polymerase produces multipleamplicons. Thus, a billion-fold isothermic amplification can be achievedusing two amplification primers.

Another version of TMA uses one primer and one or more additionalamplification oligomers to amplify nucleic acids in vitro, makingtranscripts (amplicons) that indicate the presence of the targetsequence in a sample (described in Becker et al., U.S. Pat. No.7,374,885, the details of which are hereby incorporated by referenceherein). Briefly, the single-primer TMA method uses a primer (or“priming oligomer”), a modified promoter oligomer (or“promoter-provider”) that is modified to prevent the initiation of DNAsynthesis from its 3′ end (e.g., by including a 3′-blocking moiety) and,optionally, a binding molecule (e.g., a 3′-blocked extender oligomer) toterminate elongation of a cDNA from the target strand. As referred toherein, a “T7 provider” is a blocked promoter-provider oligonucleotidethat provides an oligonucleotide sequence that is recognized by T7 RNApolymerase. This method synthesizes multiple copies of a target sequenceand includes the steps of treating a target RNA that contains a targetsequence with a priming oligomer and a binding molecule, where theprimer hybridizes to the 3′ end of the target strand. RT initiatesprimer extension from the 3′ end of the primer to produce a cDNA whichis in a duplex with the target strand (e.g., RNA:cDNA). When a bindingmolecule, such as a 3′ blocked extender oligomer, is used in thereaction, it binds to the target nucleic acid adjacent near the 5′ endof the target sequence. That is, the binding molecule binds to thetarget strand next to the 5′ end of the target sequence to be amplified.When the primer is extended by DNA polymerase activity of RT to producecDNA, the 3′ end of the cDNA is determined by the position of thebinding molecule because polymerization stops when the primer extensionproduct reaches the binding molecule bound to the target strand. Thus,the 3′ end of the cDNA is complementary to the 5′ end of the targetsequence. The RNA:cDNA duplex is separated when RNase (e.g., RNase H ofRT) degrades the RNA strand, although those skilled in the art willappreciate that any form of strand separation may be used. Then, thepromoter-provider oligomer hybridizes to the cDNA near the 3′ end of thecDNA strand. The promoter-provider oligomer includes a 5′ promotersequence for an RNA polymerase and a 3′ region complementary to asequence in the 3′ region of the cDNA. The promoter-provider oligomeralso has a modified 3′ end that includes a blocking moiety that preventsinitiation of DNA synthesis from the 3′ end of the promoter-provideroligomer. In the promoter-provider: cDNA duplex, the 3′-end of the cDNAis extended by DNA polymerase activity of RT using the promoter oligomeras a template to add a promoter sequence to the cDNA and create afunctional double-stranded promoter. An RNA polymerase specific for thepromoter sequence then binds to the functional promoter and transcribesmultiple RNA transcripts complementary to the cDNA and substantiallyidentical to the target region sequence that was amplified from theinitial target strand. The resulting amplified RNA can then cyclethrough the process again by binding the primer and serving as atemplate for further cDNA production, ultimately producing manyamplicons from the initial target nucleic acid present in the sample.Some embodiments of the single-primer transcription-associatedamplification method do not include the binding molecule and, therefore,the cDNA product made from the primer has an indeterminate 3′ end, butthe amplification steps proceed substantially as described above for allother steps.

Suitable amplification conditions can be readily determined by a skilledartisan in view of the present disclosure. “Amplification conditions” asdisclosed herein refer to conditions which permit nucleic acidamplification. Amplification conditions may, in some embodiments, beless stringent than “stringent hybridization conditions” as describedherein. Oligonucleotides used in the amplification reactions asdisclosed herein may be specific for and hybridize to their intendedtargets under amplification conditions, but in certain embodiments mayor may not hybridize under more stringent hybridization conditions. Onthe other hand, detection probes generally hybridize under stringenthybridization conditions. While the Examples section infra providespreferred amplification conditions for amplifying target nucleic acidsequences, other acceptable conditions to carry out nucleic acidamplifications could be easily ascertained by someone having ordinaryskill in the art depending on the particular method of amplificationemployed.

The amplification methods as disclosed herein, in certain embodiments,also preferably employ the use of one or more other types ofoligonucleotides that are effective for improving the sensitivity,selectivity, efficiency, etc., of the amplification reaction. These mayinclude, for example, terminating oligonucleotides, extender or helperoligonucleotides, and the like.

Target Capture

In certain embodiments, it may be preferred to purify or enrich a targetnucleic acid from a sample prior to amplification, for example using atarget capture approach. “Target capture” (TC) refers generally tocapturing a target polynucleotide onto a solid support, such asmagnetically attractable particles, wherein the solid support retainsthe target polynucleotide during one or more washing steps of the targetpolynucleotide purification procedure. In this way, the targetpolynucleotide is substantially purified prior to a subsequent nucleicacid amplification step. Numerous target capture methods are known andsuitable for use in conjunction with the methods described herein.

Any support may be used, e.g., matrices or particles free in solution,which may be made of any of a variety of materials, e.g., nylon,nitrocellulose, glass, polyacrylate, mixed polymers, polystyrene, silanepolypropylene, or metal. Illustrative examples use a support that ismagnetically attractable particles, e.g., monodisperse paramagneticbeads (uniform size.+−0.5%) to which an immobilized probe is joineddirectly (e.g., via covalent linkage, chelation, or ionic interaction)or indirectly (e.g., via a linker), where the joining is stable duringnucleic acid hybridization conditions.

For example, one illustrative approach, as described in U.S. PatentApplication Publication No 20060068417, uses at least one capture probeoligonucleotide that contains a target-complementary region and a memberof a specific binding pair that attaches the target nucleic acid to animmobilized probe on a capture support, thus forming a capture hybridthat is separated from other sample components before the target nucleicacid is released from the capture support.

In another illustrative method, Weisburg et al., in U.S. Pat. No.6,110,678, describe a method for capturing a target polynucleotide in asample onto a solid support, such as magnetically attractable particles,with an attached immobilized probe by using a capture probe and twodifferent hybridization conditions, which preferably differ intemperature only. The two hybridization conditions control the order ofhybridization, where the first hybridization conditions allowhybridization of the capture probe to the target polynucleotide, and thesecond hybridization conditions allow hybridization of the capture probeto the immobilized probe. The method may be used to detect the presenceof a target polynucleotide in a sample by detecting the captured targetpolynucleotide or amplified target polynucleotide.

Another illustrative target capture technique (U.S. Pat. No. 4,486,539)involves a hybridization sandwich technique for capturing and fordetecting the presence of a target polynucleotide. The techniqueinvolves the capture of the target polynucleotide by a probe bound to asolid support and hybridization of a detection probe to the capturedtarget polynucleotide. Detection probes not hybridized to the targetpolynucleotide are readily washed away from the solid support. Thus,remaining label is associated with the target polynucleotide initiallypresent in the sample.

Another illustrative target capture technique (U.S. Pat. No. 4,751,177)involves a method that uses a mediator polynucleotide that hybridizes toboth a target polynucleotide and to a polynucleotide fixed on a solidsupport. The mediator polynucleotide joins the target polynucleotide tothe solid support to produce a bound target. A labeled probe can behybridized to the bound target and unbound labeled pro can be washedaway from the solid support.

Yet another illustrative target capture technique is described in U.S.Pat. Nos. 4,894,324 and 5,288,609, which describe a method for detectinga target polynucleotide. The method utilizes two single-strandedpolynucleotide segments complementary to the same or opposite strands ofthe target and results in the formation of a double hybrid with thetarget polynucleotide. In one embodiment, the hybrid is captured onto asupport.

In another illustrative target capture technique, EP Pat. Pub. No. 0 370694, methods and kits for detecting nucleic acids use oligonucleotideprimers labeled with specific binding partners to immobilize primers andprimer extension products. The label specifically complexes with itsreceptor which is bound to a solid support.

The above capture techniques are illustrative only, and not limiting.Indeed, essentially any technique available to the skilled artisan maybe used provided it is effective for purifying a target nucleic acidsequence of interest prior to amplification.

Nucleic Acid Detection

Essentially any labeling and/or detection system that can be used formonitoring specific nucleic acid hybridization can be used inconjunction to detect Salmonella amplicons. Many such systems are knownand available to the skilled artisan, illustrative examples of which arebriefly discussed below.

Detection systems typically employ a detection oligonucleotide of onetype or another in order to facilitate detection of the target nucleicacid of interest. Detection may either be direct (i.e., probe hybridizeddirectly to the target) or indirect (i.e., a probe hybridized to anintermediate structure that links the probe to the target). A probe'starget sequence generally refers to the specific sequence within alarger sequence which the probe hybridizes specifically. A detectionprobe may include target-specific sequences and other sequences orstructures that contribute to the probe's three-dimensional structure,depending on whether the target sequence is present (e.g., U.S. Pat.Nos. 5,118,801, 5,312,728, 6,835,542, and 6,849,412).

Any of a number of well known labeling systems may be used to facilitatedetection. Direct joining may use covalent bonds or non-covalentinteractions (e.g., hydrogen bonding, hydrophobic or ionic interactions,and chelate or coordination complex formation) whereas indirect joiningmay use a bridging moiety or linker (e.g., via an antibody or additionaloligonucleotide(s), which amplify a detectable signal. Any detectablemoiety may be used, e.g., radionuclide, ligand such as biotin or avidin,enzyme, enzyme substrate, reactive group, chromophore such as a dye orparticle (e.g., latex or metal bead) that imparts a detectable color,luminescent compound (e.g. bioluminescent, phosphorescent orchemiluminescent compound), and fluorescent compound. Preferredembodiments include a “homogeneous detectable label” that is detectablein a homogeneous system in which bound labeled probe in a mixtureexhibits a detectable change compared to unbound labeled probe, whichallows the label to be detected without physically removing hybridizedfrom unhybridized labeled probe (e.g., U.S. Pat. Nos. 6,004,745,5,656,207 and 5,658,737). Preferred homogeneous detectable labelsinclude chemiluminescent compounds, more preferably acridinium ester(“AE”) compounds, such as standard AE or AE derivatives which are wellknown (U.S. Pat. Nos. 5,656,207, 5,658,737, and 5,948,899). Methods ofsynthesizing labels, attaching labels to nucleic acid, and detectingsignals from labels are well known (e.g., Sambrook et al., MolecularCloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989) at Chapter. 10, and U.S. Pat.Nos. 6,414,152, 5,185,439, 5,658,737, 5,656,207, 5,547,842, 5,639,604,4,581,333, and 5,731,148). Preferred methods of linking an AE compoundto a nucleic acid are known (e.g., U.S. Pat. No. 5,585,481 and U.S. Pat.No. 5,639,604, see column 10, line 6 to column 11, line 3, and Example8). Preferred AE labeling positions are a probe's central region andnear a region of A/T base pairs, at a probe's 3′ or 5′ terminus, or ator near a mismatch site with a known sequence that is the probe shouldnot detect compared to the desired target sequence.

In a preferred embodiment, oligonucleotides exhibiting at least somedegree of self-complementarity are desirable to facilitate detection ofprobe:target duplexes in a test sample without first requiring theremoval of unhybridized probe prior to detection. By way of example,when exposed to denaturing conditions, the two complementary regions ofa molecular torch, which may be fully or partially complementary, melt,leaving the target binding domain available for hybridization to atarget sequence when the predetermined hybridization assay conditionsare restored. Molecular torches are designed so that the target bindingdomain favors hybridization to the target sequence over the targetclosing domain. The target binding domain and the target closing domainof a molecular torch include interacting labels (e.g., afluorescent/quencher pair) positioned so that a different signal isproduced when the molecular torch is self-hybridized as opposed to whenthe molecular torch is hybridized to a target nucleic acid, therebypermitting detection of probe:target duplexes in a test sample in thepresence of unhybridized probe having a viable label associatedtherewith. Molecular torches are fully described in U.S. Pat. No.6,361,945, the disclosure of which is hereby incorporated by referenceherein.

Another example of a self-complementary hybridization assay probe thatmay be used is a structure commonly referred to as a “molecular beacon.”Molecular beacons comprise nucleic acid molecules having a targetcomplementary sequence, an affinity pair (or nucleic acid arms) thatholds the probe in a closed conformation in the absence of a targetnucleic acid sequence, and a label pair that interacts when the probe isin a closed conformation. Hybridization of the molecular beacon targetcomplementary sequence to the target nucleic acid separates the membersof the affinity pair, thereby shifting the probe to an openconformation. The shift to the open conformation is detectable due toreduced interaction of the label pair, which may be, for example, afluorophore and a quencher (e.g., DABCYL and EDANS). Molecular beaconsare fully described in U.S. Pat. No. 5,925,517, the disclosure of whichis hereby incorporated by reference herein. Molecular beacons useful fordetecting specific nucleic acid sequences may be created by appending toeither end of one of the probe sequences disclosed herein, a firstnucleic acid arm comprising a fluorophore and a second nucleic acid armcomprising a quencher moiety. In this configuration, Salmonella-specificprobe sequences may serve as the target-complementary “loop” portion ofthe resulting molecular beacon.

Molecular beacons are preferably labeled with an interactive pair ofdetectable labels. Preferred detectable labels interact with each otherby FRET or non-FRET energy transfer mechanisms. Fluorescence resonanceenergy transfer (FRET) involves the radiationless transmission of energyquanta from the site of absorption to the site of its utilization in themolecule or system of molecules by resonance interaction betweenchromophores, over distances considerably greater than interatomicdistances, without conversion to thermal energy, and without the donorand acceptor coming into kinetic collision. The “donor” is the moietythat initially absorbs the energy, and the “acceptor” is the moiety towhich the energy is subsequently transferred. In addition to FRET, thereare at least three other “non-FRET” energy transfer processes by whichexcitation energy can be transferred from a donor to an acceptormolecule.

When two labels are held sufficiently close such that energy emitted byone label can be received or absorbed by the second label, whether by aFRET or non-FRET mechanism, the two labels are said to be in an “energytransfer relationship.” This is the case, for example, when a molecularbeacon is maintained in the closed state by formation of a stem duplexand fluorescent emission from a fluorophore attached to one arm of themolecular beacon is quenched by a quencher moiety on the other arm.

Illustrative label moieties for the molecular beacons include afluorophore and a second moiety having fluorescence quenching properties(i.e., a “quencher”). In this embodiment, the characteristic signal islikely fluorescence of a particular wavelength, but alternatively couldbe a visible light signal. When fluorescence is involved, changes inemission are preferably due to FRET, or to radiative energy transfer ornon-FRET modes. When a molecular beacon having a pair of interactivelabels in the closed state is stimulated by an appropriate frequency oflight, a fluorescent signal is generated at a first level, which may bevery low. When this same molecular beacon is in the open state and isstimulated by an appropriate frequency of light, the fluorophore and thequencher moieties are sufficiently separated from each other such thatenergy transfer between them is substantially precluded. Under thatcondition, the quencher moiety is unable to quench the fluorescence fromthe fluorophore moiety. If the fluorophore is stimulated by light energyof an appropriate wavelength, a fluorescent signal of a second level,higher than the first level, will be generated. The difference betweenthe two levels of fluorescence is detectable and measurable. Usingfluorophore and quencher moieties in this manner, the molecular beaconis only “on” in the “open” conformation and indicates that the probe isbound to the target by emanating an easily detectable signal. Theconformational state of the probe alters the signal generated from theprobe by regulating the interaction between the label moieties.

Examples of donor/acceptor label pairs that may be used, making noattempt to distinguish FRET from non-FRET pairs, includefluorescein/tetramethylrhodamine, IAEDANS/fluorescein, EDANS/DABCYL,coumarin/DABCYL, fluorescein/fluorescein, BODIPY FL/BODIPY FL,fluorescein/DABCYL, lucifer yellow/DABCYL, BODIPY/DABCYL, eosine/DABCYL,erythrosine/DABCYL, tetramethylrhodamine/DABCYL, Texas Red/DABCYL,CY5/BH1, CY5/BH2, CY3/BH1, CY3/BH2, and fluorescein/QSY7 dye. Thosehaving an ordinary level of skill in the art will understand that whendonor and acceptor dyes are different, energy transfer can be detectedby the appearance of sensitized fluorescence of the acceptor or byquenching of donor fluorescence. When the donor and acceptor species arethe same, energy can be detected by the resulting fluorescencedepolarization. Non-fluorescent acceptors such as DABCYL and the QSY 7dyes advantageously eliminate the potential problem of backgroundfluorescence resulting from direct (i.e., non-sensitized) acceptorexcitation. Preferred fluorophore moieties that can be used as onemember of a donor-acceptor pair include fluorescein, ROX, and the CYdyes (such as CY5). Highly preferred quencher moieties that can be usedas another member of a donor-acceptor pair include DABCYL and the BlackHole Quencher moieties, which are available from Biosearch Technologies,Inc. (Novato, Calif.).

Synthetic techniques and methods of attaching labels to nucleic acidsand detecting labels are well known in the art (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; Nelson etal., U.S. Pat. No. 5,658,737; Woodhead et al., U.S. Pat. No. 5,656,207;Hogan et al., U.S. Pat. No. 5,547,842; Arnold et al., U.S. Pat. No.5,185,439 and 6,004,745; Kourilsky et al., U.S. Pat. No. 4,581,333; and,Becker et al., U.S. Pat. No. 5,731,148).

Preferred Salmonella Oligonucleotides and Oligonucleotide Sets

As described herein, preferred sites for amplifying and detectingSalmonella nucleic acids as disclosed herein have been found to residein the 350 region of Salmonella 23S rRNA. Moreover, particularlypreferred oligonucleotides and oligonucleotide sets within this regionhave been identified for amplifying Salmonella 23S with improvedsensitivity, selectivity and specificity. It will be understood that theoligonucleotides disclosed herein are capable of hybridizing to aSalmonella target sequence with high specificity and, as a result, arecapable of participating in a nucleic acid amplification reaction thatcan be used to detect the presence and/or levels of Salmonella in asample and distinguish it from the presence of other enteric bacteria.

For example, in one embodiment, the amplification oligonucleotidescomprise a first oligonucleotide and a second oligonucleotide, whereinthe first and second oligonucleotides target the 350 region of theSalmonella 23s rRNA with a high degree of specificity. Of course, itwill be understood, when discussing the amplification oligonucleotidesdisclosed herein that the first and second oligonucleotides used in anamplification reaction have specificity for opposite strands of thetarget nucleic acid sequence to be amplified.

The amplification oligonucleotides disclosed herein are particularlyeffective for amplifying a target nucleic acid sequence of Salmonella ina transcription-based amplification reaction, preferably a real-timetranscription-mediated amplification (TMA) reaction.

It will be understood that in addition to the particular T7 provideroligonucleotides and primer oligonucleotides used in the amplificationreaction, additional oligonucleotides will also generally be employed inconjunction with the amplification reaction. For example, in certainembodiments, the amplification reactions will also employ the use of oneor more of a detection oligonucleotide (e.g., a torch oligonucleotide),and a blocker oligonucleotide.

Table 1 presents specific examples of T7 Provider oligonucleotides,Primer oligonucleotides, and other ancillary oligonucleotides (e.g.,Blocker, Torch, and Target Capture oligonucleotides) that have beenidentified by the invention.

TABLE 1 Examples of Preferred Oligonucleotides SEQ ID Use NO:Sequence (5′-3′) T7 SEQ ID AATTTAATACGACTCACTATAGGGAGA- Provider NO: 1ATCAGCTTGTGTGTTAGTGGAAGC-X T7 SEQ ID AATTTAATACGACTCACTATAGGGAGA-Provider NO: 2 AGTGGAAGCGTCTGGAAAGGCGCG-X T7 SEQ IDAATTTAATACGACTCACTATAGGGAGA- Provider NO: 3 GTTAGTGGAAGCGTCTGGAAAGGC-XT7 SEQ ID AATTTAATACGACTCACTATAGGGAGA- Provider NO: 4TAGTGGAAGCGTCTGGAAAGGCGC-X T7 SEQ ID AATTTAATACGACTCACTATAGGGAGA-Provider NO: 5 GGAAGCGTCTGGAAAGGCGCGCGA-X T7 SEQ ID AATTTAATACGACTCACTATAGGGAGA- Provider NO: 6 CCAGAGCCTGAATCAGCTTGTGTG-XT7 SEQ ID  AATTTAATACGACTCACTATAGGGAGA- Provider NO: 7CGTGTGTGTTAGTGGAAGCGTCTGGAA-X T7 SEQ ID AATTTAATACGACTCACTATAGGGAGA-Provider NO: 8 CGTGTGTGTTAGTGGAAGCGTCTGGA-X T7 SEQ IDAATTTAATACGACTCACTATAGGGAGA- Provider NO: 9 CGTGTGTGTTAGTGGAAGCGTCTGG-XT7 SEQ ID AATTTAATACGACTCACTATAGGGAGA- Provider NO: 10CCACAAATCAGCTTGTGTGTTAGTGGAAGC-X T7 SEQ ID AATTTAATACGACTCACTATAGGGAGA-Provider NO: 11 CCACAACGGTTTATCAGCTTGTGTGTTAGTGG AAGC-X T7 SEQ IDAATTTAATACGACTCACTATAGGGAGA- Provider NO: 12 ATCAGCATGTGTGTTAGTGGAAGC-XT7 SEQ ID AATTTAATACGACTCACTATAGGGAGA- Provider NO: 13CCACAACGGTTTATCAGCATGTGTGTTAGTGG AAGC-X T7  SEQ IDAATTTAATACGACTCACTATAGGGAGA- Provider NO: 14 ATCAGCGTGTGTGTTAGTGGAAGC-XT7 SEQ ID AATTTAATACGACTCACTATAGGGAGA- Provider NO: 15ATCAGCTGGTGTGTTAGTGGAAGC-X T7 SEQ ID AATTTAATACGACTCACTATAGGGAGA-Provider NO: 16 CCACAACGGTTTATCAGCTGGTGTGTTAGTGG AAGC-X T7 SEQ IDAATTTAATACGACTCACTATAGGGAGA- Provider NO: 17 ATCAGCAGGTGTGTTAGTGGAAGC-XT7 SEQ ID AATTTAATACGACTCACTATAGGGAGA- Provider NO: 18CCACAACGGTTTATCAGCAGGTGTGTTAGTGG AAGC-X T7 SEQ IDAATTTAATACGACTCACTATAGGGAGA- Provider NO: 19 ATCAGCTTGTGTGTTAGTGGAAGCG-XT7 SEQ ID AATTTAATACGACTCACTATAGGGAGA- Provider NO: 20ATCAGCTTGTGTGTTAGTGGAAGCGT-X T7 SEQ ID  AATTTAATACGACTCACTATAGGGAGA-Provider NO: 21 ATCAGCTTGTGTGTTAGTGGAAGCGTC-X T7 SEQ IDAATTTAATACGACTCACTATAGGGAGA- Provider NO: 22ATCAGCTTGTGTGTTAGTGGAAGCGTCTG-X T7 SEQ ID AATTTAATACGACTCACTATAGGGAGA-Provider NO: 23 ATCAGCTTGTGTGTTAGTGGAAGCGTCTG G-X T7 SEQ IDAATTTAATACGACTCACTATAGGGAGA- Provider NO: 24ATCAGCTTGTGTGTTAGTGGAAGCGTCTG GA-X T7 SEQ IDAATTTAATACGACTCACTATAGGGAGA- Provider NO: 25ATCAGCTTGTGTGTTAGTGGAAGCGTCTGG AA-X T7 SEQ IDAATTTAATACGACTCACTATAGGGAGA- Provider NO: 26ATCAGCTTGTGTGTTAGTGGAAGCGTCT-X T7 SEQ ID AATTTAATACGACTCACTATAGGGAGA-Provider NO: 27 ATCAGCACGTGTGTTAGTGGAAGC-X T7 SEQ IDAATTTAATACGACTCACTATAGGGAGA- Provider NO: 28 ATCAGCATGCGTGTTAGTGGAAGC-XT7 SEQ ID AATTTAATACGACTCACTATAGGGAGA- Provider NO: 29ATCAGCATGTGCGTTAGTGGAAGC-X T7 SEQ ID AATTTAATACGACTCACTATAGGGAGA-Provider NO: 30 ATCAGCATGTGTGCTAGTGGAAGC-X T7 SEQ IDAATTTAATACGACTCACTATAGGGAGA- Provider NO: 31 ATCAGCATGTGTGTTAGCGGAAGC-XT7 SEQ ID AATTTAATACGACTCACTATAGGGAGA- Provider NO: 32ATCAGCAAGTGTGTTAGTGGAAGC-X T7 SEQ ID AATTTAATACGACTCACTATAGGGAGA-Provider NO: 33 CCACAAATCAGCTTGTGTGTTAGTGGAAGC GTCT-X T7 SEQ IDAATTTAATACGACTCACTATAGGGAGA- Provider NO: 34CCACAACGGTTTATCAGCTTGTGTGTTAGT GGAAGCGTCT-X Primer SEQ IDTCACAGCACATGCGC NO: 35 Primer SEQ ID CTCACAGCACATGCGC NO: 36 PrimerSEQ ID GCTCACAGCACATGCGC NO: 37 Primer SEQ ID AGCTCACAGCACATGCGC NO: 38Primer SEQ ID AGCTCACAGCACATcCGC NO: 39 Primer SEQ IDCGAGCTCACAGCACATGCGC NO: 40 Primer SEQ ID cgagCTCACAGCACATGCGC NO: 41Primer SEQ ID cgagCTCACAGCACATCCGC NO: 42 Primer SEQ IDATCGAGCTCACAGCACATGCGC NO: 43 Primer SEQ ID aucgAGCTCACAGCACATGCGCNO: 44 Primer SEQ ID aucgAGCTCACAGCACATCCGC NO: 45 Primer SEQ IDacucATCGAGCTCACAGCACATGCGCT NO: 46 Primer SEQ ID CGAGCTCACAGCACATCCGCNO: 47 Primer SEQ ID ATCGAGCTCACAGCACATCCGC NO: 48 Primer SEQ IDAGCTCACAGCAGATCCGC NO: 49 Primer SEQ ID AGCTCACAGCACCTCCGC NO: 50 PrimerSEQ ID AGCTCACAGCAGCTCCGC NO: 51 Primer SEQ IDGCTCACAGCACATGCGCTTTTGTGTACG NO: 52 Primer SEQ IDCTCATCGAGCTCACAGCACATGCGCTTTTGTG NO: 53 Primer SEQ IDCCCTACTCATCGAGCTCACAGCAC NO: 54 Primer SEQ ID GGATACCACGTGTCCCGCCCTACTCNO: 55 Primer SEQ ID CGAGCTCACAGCACATGCGCTTTTGTGTACG NO: 56 PrimerSEQ ID AGCTCACAGCACATGCCC NO: 57 Primer SEQ IDCGAGCTCACAGCACACGCGCTTTTGTGTACG NO: 58 Blocker SEQ IDcugauucaggcucugggcucc-X NO: 59 Blocker SEQ ID ccacuaacacacacgcugau-XNO: 60 Blocker SEQ ID cuaacacacacgcugauucagg-X NO: 61 Blocker SEQ IDcacuaacacacacgcugauucagg-X NO: 62 Blocker SEQ ID cuuccacuaacacacacgcuNO: 63 Blocker SEQ ID ucugggcuccuccccguucg NO: 64 Blocker SEQ IDacacgcugauucaggcucugg-X NO: 65 Torch SEQ ID ggcugucacccuguau9cagccNO: 66 Torch SEQ ID cgcgc9ugucacccuguaucgcgcg NO: 67 Torch SEQ IDcacccuguaucgcgc9gggug NO: 68 Torch SEQ ID cacccuguaucgcgcgccuuuc9gggugNO: 69 Torch SEQ ID cccc9gcuuuuguguacgggg NO: 70 Target SEQ IDccgguucgccucauuaacc- Capture NO: 71 TTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATarget SEQ ID ccucgggguacuuagauguuuc- Capture NO: 72TTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Target SEQ IDggaaucucgguugauuucuuuucc- Capture NO: 73TTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Target SEQ ID ccguucgcucgccgcuacug-Capture NO: 74 TTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Target SEQ IDcugauucaggcucugggcucc- Capture NO: 75 TTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATarget SEQ ID cagacaggataccacgtgtcc- Capture NO: 76TTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Target SEQ ID cccatattcagacaggatacc-Capture NO: 77 TTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Lower case 2′-O--methylRNA X is a blocking moiety (e.g., reverse(3′-5′) C blocked) 9 is anon-nucleotide (e.g.,triethylene glycol) linker joining region, and5′-fluorescein (“F”) fluorophore and 3′-dabsyl (“D”) quencher moietieswere attached to the torch oligonucleotides

In addition, Table 2 identifies two particularly preferredoligonucleotide sets for use in the compositions, kits and methods asdisclosed herein.

TABLE 2 Example of Two Preferred Oligonucleotide Sets OligonucleotideSet Description Oligonucleotide Set #1 T7 Provider SEQ ID NO: 17 BlockerSEQ ID NO: 59 Primer SEQ ID NO: 50 Torch SEQ ID NO: 66 Set #2 T7Provider SEQ ID NO: 26 Blocker SEQ ID NO: 59 Primer SEQ ID NO: 49 TorchSEQ ID NO: 66

While specifically preferred amplification oligonucleotides derived fromthe 350 region have been identified, which result in superior assayperformance, it will be recognized that other oligonucleotides derivedfrom the 350 region and having insubstantial modifications from thosespecifically described herein may also be used, provided the same orsimilar performance objectives are achieved. For example,oligonucleotides derived from the 350 region and useful in theamplification reactions as disclosed herein can have different lengthsfrom those identified herein, provided it does not substantially affectamplification and/or detection procedures. These and other routine andinsubstantial modifications to the preferred oligonucleotides can becarried out using conventional techniques, and to the extent suchmodifications maintain one or more advantages provided herein they areconsidered within the spirit and scope of the invention.

The general principles as disclosed herein may be more fully appreciatedby reference to the following non-limiting Examples.

EXAMPLES

Examples are provided below illustrating certain aspects andembodiments. The examples below are believed to accurately reflect thedetails of experiments actually performed, however, it is possible thatsome minor discrepancies may exist between the work actually performedand the experimental details set forth below which do not affect theconclusions of these experiments or the ability of skilled artisans topractice them. Skilled artisans will appreciate that these examples arenot intended to limit the invention to the specific embodimentsdescribed therein. Additionally, those skilled in the art, using thetechniques, materials and methods described herein, could easily deviseand optimize alternative amplification systems for carrying out theseand related methods while still being within the spirit and scope of thepresent invention.

Unless otherwise indicated, oligonucleotides and modifiedoligonucleotides in the following examples were synthesized usingstandard phosphoramidite chemistry, various methods of which are wellknown in the art. See e.g., Carruthers, et al., 154 Methods inEnzymology, 287 (1987), the contents of which are hereby incorporated byreference herein. Unless otherwise stated herein, modified nucleotideswere 2′-O-methyl ribonucleotides, which were used in the synthesis astheir phosphoramidite analogs. For blocked oligonucleotides used insingle-primer amplification (Becker et al., U.S. Pat. No. 7,374,885,hereby incorporated by reference herein), the 3′-terminal blockingmoiety consisted of a “reversed C” 3′-to-3′ linkage prepared using3′-dimethyltrityl-N-benzoyl-2′-deoxycytidine, 5′-succinoyl-long chainalkylamino-CPG (Glen Research Corporation, Cat. No. 20-0102-01).Molecular torches (see Becker et al., U.S. Pat. No. 6,849,412, herebyincorporated by reference herein) were prepared using a C9non-nucleotide (triethylene glycol) linker joining region (SpacerPhosphoramidite 9, Glen Research Corporation, Cat. No. 10-1909-xx),5′-fluorescein (“F”) fluorophore and 3′-dabsyl (“D”) quencher moietiesattached to the oligonucleotide by standard methods.

As set forth in the examples below, analyses of a wide variety ofamplification reagents and conditions has led to the development of ahighly sensitive and selective amplification process for the detectionof Salmonella. The raw real-time amplification assay charts of multiplereplicates of analyte at different target concentrations (see, e.g.,FIG. 1) were utilized to assess the quality of the oligonucleotide sets.The data from the real-time assays were collected and analyzed tocalculate TTime values and RFU range for presentation of data hereinbelow.

Example 1 Description of Illustrative Assay Reagents and Protocols

The following example describes typical assay reagents, protocols,conditions and the like used in the real-time TMA experiments describedherein. Unless

A. Reagents and Samples

1. Amplification Reagent. The “Amplification Reagent” or “Amp Reagent”comprised approximate concentrations of the following components: 0.5 mMdATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP, 10 mM ATP, 2 mM CTP, 2 mMGTP, 12.7 mM UTP, 30 mM MgCl₂, and 33 mM KCl in 50 mM HEPES buffer at pH7.7. Primers and other oligonucleotides were added to the Amp Reagent.

2. Enzyme Reagent. The “Enzyme Reagent” comprised approximateconcentrations of the following components: 1180 RTU/μL Moloney murineleukemia virus (“MMLV”) reverse transcriptase (“RT”) and 260 PU/μL T7RNA polymerase in 75 mM HEPES buffer containing 120 mM KCl, 10% TRITON®X-100, 160 mM N-acetyl-L-cysteine, and 1 mM EDTA at pH 7.0, where oneRTU of RT activity incorporates 1 nmol of dT into a substrate in 20minutes at 37° C. and one PU of T7 RNA polymerase activity produces 5fmol of RNA transcript in 20 minutes at 37° C.

3. Wash Solution. The “Wash Solution” comprised 0.1% (w/v) sodiumdodecyl sulfate, 150 mM NaCl and 1 mM EDTA in 10 mM HEPES buffer at pHto 7.5.

4. Target Capture Reagent. The “Target Capture Reagent” (TCR) comprisedapproximate concentrations of the following components: 60 pmol/mL eachof one or more capture probes having a dT₃dA₃₀ tail and an optionalcapture helper probe, 250 to 300 ug/mL paramagnetic oligo-(dT)₁₄microparticles (Seradyn), 250 mM HEPES, 100 mM EDTA and 1.88 M LiCl atpH 6.5.

5. Lysis Reagent. The “Lysis Buffer” comprised 1% lithium lauryl sulfatein a buffer containing 100 mM tris, 2.5 mM succinic acid, 10 mM EDTA and500 mM LiCl at pH 6.5.

6. Target rRNA Samples. rRNA samples were stored in water, 0.1% LiLS orLysis Reagent prior to use in the experiments described herein.

B. Equipment and Material

-   -   KingFisher® 96 (Thermo Electron, Waltham, Mass.)    -   FLUOstar (BMG LABTECH, Germany)    -   eppendorf® Thermomixer R 022670565 (Eppendorf Corporation,        Westbury, N.Y.)    -   Hard-Shell Thin-Wall 96-Well Skirted PCR Plates, colored        shell/white well, Catalog numbers: HSP-9615, HSP-9625, HSP-9635)        (BioRad Hercules, Calif.)    -   KingFisher® 96 tip comb for DW magnets (Catalog        number: 97002534) Thermo Electron, Waltham, Mass.)    -   DW 96 plate, V bottom, Polypropylene, sterile 25 pcs/case        (Axygen Catalog number: P-2ML-SQ-C-S; VWR catalog number        47749-874)    -   KingFisher® 96 KF plate (200 microlitres) (Catalog number:        97002540)    -   PTI® plate reader    -   Chromo4™ plate reader        C. Target Capture

Samples were mixed with Lysis Reagent to release target and stabilizerRNA. Target Capture Reagent was added. Ribosomal RNA target wascaptured and purified on magnetic particles using the KingFisher 96purification system. Particles were resuspended in Amplification Reagentcontaining FAM-labeled Torch for analyte and TAMRA-labeled Torch for theinternal control. A typical target capture procedure to purify andprepare nucleic acid samples for subsequent amplification was performedessentially as described below. 100 μL of test sample, 50 μL of the TCRcontaining target capture oligonucleotides, and 1 mL Lysis Reagent werecombined and incubated at 60° C. for 15 minutes. The TCR magneticparticles from the treated reaction mixture were captured and washedusing the Wash Solution and a suitable magnetic particle washing andseparation device (e.g., a magnetic separation rack, a GEN-PROBE® TargetCapture System, Gen-Probe Cat. No. 5207, or a KingFisher® magneticparticle processor system available from Thermo Labsystems). Afterwashing, the magnetic particles were resuspended in 100 μL of theAmplification Reagent.

D. Amplification and Detection of Target

The real-time TMA amplification reactions were performed essentially asfollows. 30 μL of sample, amplification and detection oligonucleotidesin the Amp Reagent or 30 μL of the resuspended particles in the AmpReagent from the target capture procedure was incubated at 60° C. for 10minutes. The temperature was then reduced and the reaction mixture wasequilibrated to 42° C. on an Eppendorf Thermomixer incubator for 15minutes. 10 μL of Enzyme Reagent was added. The reaction mixture wasmixed and incubated for 75 minutes at 42° C. in a real-time detectionsystem (e.g., Opticon™ or Chromo4™ detection systems available fromBio-Rad Laboratories, or a PTI FluoDia® T70 instrument) for simultaneousamplification and detection of analyte and the internal control.

Example 2 Design and Initial Testing of Salmonella Oligonucleotide Sets

Using a region corresponding to the 350 region of the E. coli rRNAsequence, several T7 Providers, Blockers, Primers, and Torches weredesigned. This region was selected because it contains mismatches thatare unique to other non-Salmonella enteric bacteria.

A total of 426 sets of T7, Blocker, Primer and Torch oligonucleotideswere screened using a plate screening protocol. The SEQ ID NOs: ofpreferred oligonucleotides are given in Table 3. The number ofoligonucleotides and concentrations used were: 8 different T7s (5pmol/rxn); 7 different Blockers (0.5 pmol/rxn); 12 different Primers (5pmol/rxn) and 5 different Torches (8 pmol/rxn). The target used wasSalmonella enterica ssp. enterica sv. Enteritidis (ATCC 13076/GP60) rRNAat 1E+4 copies per rxn. The raw data collected were analyzed tocalculate TTime values and RFU range. The data derived were grouped intosets giving TTime below 30 min, between 30 to 35 min, and those thatwere 35 to 39 min (Table 4).

TABLE 3 Oligonucleotides Used for Screening 23S “350” Region Use SEQ IDNOs: T7 Providers SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8 Primers SEQ ID NOs:35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 Torches SEQ ID NOs: 66,67, 68, 69, 70 Blockers SEQ ID NOs: 59, 60, 61, 62, 63, 64, 65

TABLE 4 RFU and TTime Values Oligonucletide Combination SEQ ID Nos: ofProvider:Blocker:Primer:Torch RFU Range TTime SEQ ID NOs: 6:64:46:6633768 22.86 SEQ ID NOs: 6:64:46:68 14696 26.43 SEQ ID NOs: 6:64:43:6626036 26.62 SEQ ID NOs: 3:61:38:70 19878 27.22 SEQ ID NOs: 3:61:46:7025349 29.58 SEQ ID NOs: 3:61:42:66 27285 29.64 SEQ ID NOs: 6:64:46:6910330 29.85 SEQ ID NOs: 6:64:42:66 29060 29.92 SEQ ID NOs: 6:64:42:6726651 29.92 SEQ ID NOs: 3:61:39:68 19129 30.13 SEQ ID NOs: 6:64:42:6818237 30.18 SEQ ID NOs: 1:59:46:68 8391 30.52 SEQ ID NOs: 6:64:43:6818629 30.53 SEQ ID NOs: 3:61:39:66 27392 30.81 SEQ ID NOs: 1:59:39:6626912 30.94 SEQ ID NOs: 1:59:46:66 25411 31.00 SEQ ID NOs: 6:64:42:7033095 31.29 SEQ ID NOs: 1:59:46:67 25661 31.37 SEQ ID NOs: 1:59:42:6734611 31.41 SEQ ID NOs: 1:59:46:70 16521 31.74 SEQ ID NOs: 3:61:46:6822371 31.94 SEQ ID NOs: 3:61:42:70 24300 31.94 SEQ ID NOs: 1:59:39:6813141 32.03 SEQ ID NOs: 3:61:39:70 19397 32.88 SEQ ID NOs: 1:59:42:6812664 33.15 SEQ ID NOs: 1:59:45:70 16806 33.47 SEQ ID NOs: 6:64:38:6618289 34.17 SEQ ID NOs: 1:59:39:70 18714 34.21 SEQ ID NOs: 6:64:42:699186 34.59 SEQ ID NOs: 6:64:38:68 18537 34.80 SEQ ID NOs: 1:59:38:6817840 34.83 SEQ ID NOs: 1:59:39:67 18239 34.83 SEQ ID NOs: 1:59:45:6730804 34.92 SEQ ID NOs: 6:64:45:68 18529 35.01 SEQ ID NOs: 3:61:39:6721912 35.33 SEQ ID NOs: 1:59:45:68 9103 35.92 SEQ ID NOs: 1:59:38:7018450 36.41 SEQ ID NOs: 1:59:45:66 13572 37.68 SEQ ID NOs: 1:59:37:6913248 38.14 SEQ ID NOs: 3:61:35:68 20426 38.21 SEQ ID NOs: 1:59:38:6715171 38.29 SEQ ID NOs: 6:64:40:68 18404 38.31 SEQ ID NOs: 1:59:40:7011004 38.54 SEQ ID NOs: 5:63:42:66 26120 38.72 SEQ ID NOs: 3:61:35:6717633 38.84 SEQ ID NOs: 6:64:45:70 27222 38.94 SEQ ID NOs: 1:59:43:6716832 39.07 SEQ ID NOs: 6:64:38:67 18945 39.32 SEQ ID NOs: 6:64:38:7015589 39.35

A secondary screening was performed on 42 potential oligonucleotide setsbased on the initial screening. The oligonucleotides used are shown inTable 5.

TABLE 5 Oligonucleotides Used for Secondary Screening Use SEQ ID NOs: T7Providers SEQ ID NOs: 1, 3, 5, 6 Primers SEQ ID NOs: 35, 37, 38, 39, 40,42, 43, 45, 46 Torches SEQ ID NOs: 66, 67, 68, 69, 70 Blockers SEQ IDNOs: 59, 61, 63, 64

In addition to repeating the reactivity to S. Enteritidis (GP60) rRNA, apreliminary cross-reactivity test against E. coli (GP88/ATCC10798) rRNAwas also performed. From these results, 4 sets were identified thateither did not cross-react with E. coli or did cross-react with E. colibut with a lag in the emergence time. These 4 oligonucleotide sets areshown in Table 6.

TABLE 6 Oligonucleotides Used for Additional Screening OligonucleotideSet Description Oligonucleotide Set #3 T7 Provider SEQ ID NO: 1 BlockerSEQ ID NO: 59 Primer SEQ ID NO: 39 Torch SEQ ID NO: 66 Set #4 T7Provider SEQ ID NO: 1 Blocker SEQ ID NO: 59 Primer SEQ ID NO: 43 TorchSEQ ID NO: 66 Set #5 T7 Provider SEQ ID NO: 6 Blocker SEQ ID NO: 64Primer SEQ ID NO: 45 Torch SEQ ID NO: 66 Set #6 T7 Provider SEQ ID NO: 6Blocker SEQ ID NO: 64 Primer SEQ ID NO: 42 Torch SEQ ID NO: 67

Initial specificity testing against other enteric bacteria (namely,Enterobacter cloacae and Citrobacter freundii) was performed for all 4oligonucleotide sets. From these 4 oligonucleotide sets, the bestoligonucleotide set was identified as Set #3 because it did notcross-react with E. coli (GP88), E. cloacae and C. freundii. It also diddetect Salmonella bongori, the other species under the genus Salmonella.Repeat testing of Salmonella 23S oligonucleotide Set #3 was done withmore replicates of S. Enteritidis, S. bongori, C. freundii, E. cloacaeand 3 E. coli strains (GP3/ATCC25922, GP88/ATCC10798, andGP831/ATCC29214). S. Enteritidis and S. bongori rRNAs were againdetected to a level similar to what was obtained previously. However,one E. coli strain (GP831) was also detected by this set ofoligonucleotides.

Alternative Regions

Designs for the Salmonella genus project were started in the 450 regionof the 16S rRNA (Table 7). Sequences were screened in that same manneras those in the 23S rRNA discussed above. Designs for the assay focusedon the mismatches shown between bases 450-490. It was shown that theCitrobacter and Enterobacter strains were very close if not identical tothe Salmonella in this region. It was also determined that S. bongoriand S. arizonae were more similar to E. coli than other Salmonella andposed the risk of false negative generation. Initial screening resultsshowed the inability of the 16S oligonucleotide system to discriminatethe Citrobacter and Enterobacter strains tested. The data showed a veryhigh false positivity rate that was inherent to the system. Based oninitial screening results, it was decided to move forward with alternatedesigns (23S-350 region) since Enterobacter and Citrobacter could not bediscriminated.

TABLE 7 Oligonucleotides Used for Screening 16S “450” Region SEQ ID UseNO: Sequence Blocker SEQ ID  gcggcauggcugcauccgga NO: 78 Blocker SEQ ID cauacacgcggcauggcugc-X NO: 79 Blocker SEQ ID  uucauacacgcggcauggcu-XNO: 80 Blocker SEQ ID  ccuucuucauacacgcggca-X NO: 81 Blocker SEQ ID cuucuucauacacgcg-X NO: 82 Blocker SEQ ID  gccuucuucauacacgcg-X NO: 83Blocker SEQ ID  aggccuucuucauacacgcg-X NO: 84 Blocker SEQ ID gaaggccuucuucauacacg NO: 85 Blocker SEQ ID  gaaggccuucuucauacacg-XNO: 86 Blocker SEQ ID  caacccgaaggccuucuuc-X NO: 87 Blocker SEQ ID aguacuuuacaacccgaagg NO: 88 Blocker SEQ ID  cgcugaaaguacuuuacaac NO: 89T7 SEQ ID ATTTAATACGACTCACTATAGGGAGAGCCG Provider NO: 90CGTGTATGAAGAAGGCCTTC-X T7 SEQ ID AATTTAATACGACTCACTATAGGGAGAGTG ProviderNO: 91 TATGAAGAAGGCCTTCGGGTTGTAAAG-X T7 SEQ IDATTTAATACGACTCACTATAGGGAGAATGA Provider NO: 92 AGAAGGCCTTCGGGTTGTAAAG-XT7 SEQ ID ATTTAATACGACTCACTATAGGGAGAGAAG Provider NO: 93GCCTTCGGGTTGTAAAG-X T7 SEQ ID ATTTAATACGACTCACTATAGGGAGACCAC ProviderNO: 94 AAGAAGGCCTTCGGGTTGTAAAG-X T7 SEQ IDATTTAATACGACTCACTATAGGGAGAGAAG Provider NO: 95 GCCTTCGGGTTGTAAAGTA-X T7SEQ ID ATTTAATACGACTCACTATAGGGAGAGAAG Provider NO: 96GCCTTCGGGTTGTAAAGTACTT-X T7 SEQ ID AATTTAATACGACTCACTATAGGGAGAGGCProvider NO: 97 CTTCGGGTTGTAAAGTACTTTCAGCGG-X T7 SEQ IDATTTAATACGACTCACTATAGGGAGACCTT Provider NO: 98 CGGGTTGTAAAGTACTTTC-X T7SEQ ID ATTTAATACGACTCACTATAGGGAGAGGGT Provider NO: 99TGTAAAGTACTTTCAGCGG-X T7 SEQ ID AATTTAATACGACTCACTATAGGGAGAGTT ProviderNO: 100 GTAAAGTACTTTCAGCGGGGAGGAAGG-X T7 SEQ IDATTTAATACGACTCACTATAGGGAGAGTAC Provider NO: 101 TTTCAGCGGGGAGGAAGG-X T7SEQ ID ATTTAATACGACTCACTATAGGGAGAGTAC Provider NO: 102TTTCAGCGGGGAGGAAGG-X T7 SEQ ID ATTTAATACGACTCACTATAGGGAGAGTAC ProviderNO: 103 TTTCAGCGGGGAGGAAGGGAGTAAAG-X T7 SEQ IDATTTAATACGACTCACTATAGGGAGACAGC Provider NO: 104 GGGGAGGAAGGGAGTAAAG-XExtender SEQ ID TACTTTCAGCGGGGAGGAAGG NO: 105 Extender SEQ IDTACTTTCAGCGGGGAGGAAGGGAG NO: 106 Primer SEQ ID CGGGTTGTAAAGTACTTTCAGCGGNO: 107 Primer SEQ ID GAACCTAGTTGGGCGAGTTACGGA NO: 108GTAACGTCAATTGCTGCGGT Primer SEQ ID GTAACGTCAATTGCTGCGGT NO: 109 PrimerSEQ ID GGTAACGTCAATTGCTGCGG NO: 110 Primer SEQ IDGAACCTAGTTGGGCGAGTTACGGA NO: 111 GGTAACGTCAATTGCTGCGG Primer SEQ IDCTGCGGGTAACGTCAATTGCTG NO: 112 Primer SEQ ID GAACCTAGTTGGGCGAGTTACGGANO: 113 CTGCGGGTAACGTCAATTGCTG Primer SEQ IDGTTTGTATGTCTGTTGCTATTATGTCTACC NO: 114 TTCTTCTGCGGGTAACGTCAATG PrimerSEQ ID cacgGAGTTAGCCGGTGCTTC NO: 115 Primer SEQ IDcugcTGGCACGGAGTTAGCCGGTGCTTC NO: 116 Primer SEQ IDGTTTGTATGTCTGTTGCTATTATGTCTACC NO: 117 TGCTGGCACGGAGTTAGCCGGTGCTTCPrimer SEQ ID GTCTACGCGGCTGCTGGCACGGAGTTAGCC NO: 118 GGTGCTTC PrimerSEQ ID GAACCTAGTTGGGCGAGTTACGGA NO: 119 GTCTACGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTC Primer SEQ ID cugcTGGCACGGAGTTAGC NO: 120 Primer SEQ IDcgcuTGCACCCTCCGTATTACCGCGGC NO: 121 Primer SEQ ID cgcuTGCACCCTCCGTATTACCNO: 122 Primer SEQ ID GTTTGTATGTCTGTTGCTATTATGTCTACG NO: 123GAUTTCACATCTGACTTAACAAAC Torch SEQ ID ggggcuuuacucccuuccucccc NO: 124Torch SEQ ID ggagg9accacaacaccuuccucc NO: 125 Torch SEQ IDggagg9uuauuaaccacaacaccuuccucc NO: 126 Torch SEQ IDcgagg9accacaacaccuuccucg NO: 127 Torch SEQ ID ccaacuuuacucccuuccucguuggNO: 128 Torch SEQ ID gcaaagguauuaacuuuacucccuuccuuu NO: 129 gc TorchSEQ ID ggaagg9uuauuaaccacaacaccuucc NO: 130 Torch SEQ IDgcaaagguauuaacuuuacucccuuugc NO: 131 Torch SEQ IDgggaggguauuaacuuuacuccc NO: 132 Torch SEQ ID cggug9uuauuaaccacaacaccgNO: 133 Torch SEQ ID gguguu9auuaaccacaacacc NO: 134 Torch SEQ IDcggug9gcugcgguuauuaaccacaacacc NO: 135 g Torch SEQ IDcgcugcgguuauuaaccacaa9cagcg NO: 136 Torch SEQ IDgcugcgguuauuaaccacaaca9gcagc NO: 137 Torch SEQ IDccugcugcgguuauuaaccacaaca9gcag NO: 138 g Torch SEQ IDcgaggagcaaagguauuaacuuuacucgg NO: 139 Torch SEQ IDcgagcaaagguauuaacuuuacucgcucg NO: 140 Torch SEQ IDcgagcaaagguauuaacuuuacugcucg NO: 141 Torch SEQ IDcgagcaaagguauuaacuuuacgcucg NO: 142 Torch SEQ IDcgagcaaagguauuaacuuuagcucg NO: 143 Torch SEQ IDcgagcaaagguauuaacuuugcucg NO: 144 Torch SEQ ID cgagcaaagguauuaacgcucgNO: 145 Torch SEQ ID ccgucaaugagcaaaggacgg NO: 146 Torch SEQ IDcggguaacgucaaugagcaaaggacccg NO: 147 Torch SEQ IDcugcggguaacgucaaugagcaaacgcag NO: 148 Torch SEQ IDccugcggguaacgucaaugagcagg NO: 149 Lower case 2′-O--methyl RNA Xis ablocking moiety (e.g., reverse(3′-5′) C blocked) 9 is a non-nucleotide(triethylene glycol) linker joining region, and 5′-fluorescein (“F”)fluorophore and 3′-dabsyl (“D”) quencher moieties were attached to thetorch oligonucleotides

Accordingly, the 350 region of the 23S rRNA was selected as thepreferred region for further optimization based upon the finding that T7providers and primer oligonucleotides for this region displayed thehighest signals and lowest background in a single primer TMA assay,relative to the large number of other oligonucleotide sets tested.Screening of oligonucleotides in a TMA assay was performed, anddifferent Torches and Blockers were also analyzed. The criteria forselecting the best oligonucleotide sets included having the lowestbackground and the highest signal at 1E+5 copies of Salmonella rRNA.

Example 3 Further Identification of Salmonella Oligonucleotide Sets

To further reduce background signals and improve specificity andsensitivity, a number of additional oligonucleotide sets were designedand tested.

Based on oligonucleotide set #3 several redesigned T7 providers andprimer oligonucleotides were identified that took advantage ofmismatches found in E. coli. Real-time TMA was run on the redesigned 23ST7 providers and primer oligonucleotides. The number of oligonucleotidesand concentrations used were: 10 different T7 Providers (5pmol/reaction); 1 Blocker (0.5 pmol/reaction); 6 Primer oligonucleotides(5 pmol/reaction) and 1 Torch oligonucleotide (8 pmol/reaction). Theidentities of the oligonucleotides are shown in Table 8.

TABLE 8 Redesigned Oligonucleotides Use SEQ ID NO: T7 Providers SEQ IDNOs: 1, 10, 11, 12, 13, 14, 15, 16, 17, 18 Primers SEQ ID NOs: 39, 40,43, 47, 48, 49 Blocker SEQ ID NO: 59 Torch SEQ ID NO: 66

The targets used were S. Enteritidis GP60 rRNA at 1E+4 copies perreaction, S. bongori at 1E+4 copies per reaction, E. coli GP88 rRNA at1E+6 copies/rxn and E. coli GP831 rRNA at 1E+6 copies/rxn. A total of 60sets were tested and 10 potential oligonucleotide sets from thisredesigned set were identified to give respectable TTimes and RFU rangesfor S. Enteritidis, but some did not react with S. bongori or didcross-react with E. coli GP88 and/or E. coli GP831 (Table 9).

TABLE 9 TTime and RFU range of 10 Potential Oligonucleotide Sets OLIGOCOMBINATION Provider:Blocker:Primer:Torch (SEQ ID NOs:) Target> SE at10{circumflex over ( )}4 SB at 10{circumflex over ( )}4 EC0088 at10{circumflex over ( )}6 EC0831 at 10{circumflex over ( )}6 SEQ ID NOs:1:59:39:66 TTIME 29.49 30.16    0 37.44 RFU 34,713 1,113    0 22,417 SEQID NOs: 12:59:47:66 TIME 34.11 40.06    0 40.19 RFU 30,963 3.357    014,759 SEQ ID NOs: 12:59:49:66 TTIME 28.67 30.53    30.91* 34.19 RFU32,554 15.103   1145* 26,336 SEQ ID NOs: 13:59:47:66 TTIME 32.17 44.14   0 40.37 RFU 29.643 3.037    0 2,541 SEQ ID NOs: 13:59:48:66 TTIME28.42 38.55    44.91 36.4 RFU 28,184 11,519  1,534 4,687 SEQ ID NOs:14:59:49:66 TTIME 27.5 33.24    0 39.29 RFU 27,505 14,480    0 18,138SEQ ID NOs: 15:59:49:66 TTIME 30.83 32.22    43.11 46.27 RFU 35.5641,766  1,014 19,568 SEQ ID NOs: 17:59:48:66 TTIME 33.96 34.24    46.01**44.58 RFU 22.283 1,854   2018** 19.210 SEQ ID NOs: 17:59:49:66 TTIME30.46 30.7    46.12 42.08 RFU 27,194 25,054 21,684 14,927 SEQ ID NOs:10:59:48:66 TTIME 35.56 43.06    52.25 45.31 RFU 29,914 20,081  1,32710,999 *0 target had lower TTime and same RFU; **0 target had higherTtime and lower RFU Lower TTime and higher TTime refer to earlier andlater emergence of signal, respectively.

Real-time TMA was used to screen a subset of the above 10 sets using thefollowing concentrations: T7 Providers at 5 pmol/reaction; Blockeroligonucleotide at 0.5 pmol/rxn; Primer oligonucleotides at 5pmol/reaction and Torch oligonucleotide at 8 pmol/rxn. The targets usedwere S. Enteritidus rRNA at 1E+4 cps/rxn, S. bongori at 1E+4 cps/rxn; 13strains of E. coli at 1E+7 cps/rxn; C. freundii at 1E+6 cps/rxn; E.cloacae at 1E+6 cps/rxn; 2 strains of Shigella flexneri at 1E+6 cps/rxn,and Shigella sonnei at 1E+6 cps/rxn.

The T7 Provider and primer oligonucleotides were mixed and matched toprovide 4 possible sets to test that are shown in Table 10.

TABLE 10 Oligonucleotide Sets Oligonucleotide Set DescriptionOligonucleotide Set 3 T7 Provider SEQ ID NO: 1 Blocker SEQ ID NO: 59Primer SEQ ID NO: 39 Torch SEQ ID NO: 66 Set 7 T7 Provider SEQ ID NO: 1Blocker SEQ ID NO: 59 Primer SEQ ID NO: 49 Torch SEQ ID NO: 66 Set 8 T7Provider SEQ ID NO: 17 Blocker SEQ ID NO: 59 Primer SEQ ID NO: 39 TorchSEQ ID NO: 66 Set 9 T7 Provider SEQ ID NO: 17 Blocker SEQ ID NO: 59Primer SEQ ID NO: 49 Torch SEQ ID NO: 66

These sets were tested against all 13 strains of E. coli (ATCC#'s 25922,11775, 10798, 35150, 33780, 23722, 25404, 29214, 29194, 35359, 23499,12792, and 23503), then against other enteric bacteria. The TTime andRFU results were compared to each other. Using the best 3oligonucleotide sets obtained from the E. coli results, real-time TMAwas run on several other enteric bacteria using sets #3, #7, and #9. All3 oligonucleotide sets picked up S. enterica with TTimes of 28 min, 25and 23 min, respectively. S. bongori was also picked up by these 3oligonucleotide sets with TTimes of 32 min, 30 min and 26 min,respectively. Some other enteric bacteria showed some cross-reactivity,but had very late emergence times and low RFU levels.

Based on the results obtained, further assay testing and optimizationfocused on 2 oligonucleotide sets: #7 and #9 (Table 10). Sensitivity ofdetecting various copy levels of S. Enteritidis GP60 rRNAs in a puresystem (no target capture step) using oligonucleotide sets #7 and #9 wastested. At 1E+5 copies, TTime was in the low 20 min range for both setsof oligonucleotides. Oligonucleotide set #7 detected 83% of replicatesat the 100-copy level. Oligonucleotide set #9 detected 100% ofreplicates at the 50-copy level.

Example 4 Further Characterization and Optimization of SalmonellaOligonucleotide Sets

Based on the results of set #7 showing better specificity than set #9and of set #9 showing better sensitivity than set #7, newoligonucleotide redesigns of both T7 provider and primeroligonucleotides were prepared.

Oligonucleotide Set #7

Newly redesigned T7 oligonucleotide providers were tested and comparedto the original T7 Provider sequence of SEQ ID NO: 1, which, incombination with the Blocker sequence of SEQ ID NO: 59, the primersequence of SEQ ID NO: 49, and the Torch sequence of SEQ ID NO: 66,provided the least cross-reactivity to E. coli. Amplificationperformance was evaluated for each set of oligonucleotides compared toset #7. The targets all went through Target Capture step using thesequence of SEQ ID NO: 74. From the data and shape of the curves, T7provider sequences of SEQ ID NOs: 24 and 26 were selected to be furtherevaluated.

Oligonucleotide Set #9

Primer oligonucleotides of SEQ ID NOs: 50 and 51 were redesigned fromthe sequence of SEQ ID NO: 49 to take advantage of other possiblemismatches to E. coli and in order to reduce cross-reactivity of thesequence of SEQ ID NO: 49 to E. coli. Testing was done without targetcapture to establish baseline performance measurement.

Table 11 presents redesigned oligonucleotides. The redesignedoligonucleotides had the lowest relative fluorescence unit (RFU) and thelongest TTime at the zero rRNA copy level. High RFU values at the zerorRNA copy level indicated possible contamination within the reagents.

TABLE 11 Redesigned Oligonucleotides Use SEQ ID NO: T7 Provider SEQ IDNOs: 19, 20, 21, 22, 23, 24, 25, 26 Primer SEQ ID NOs: 50, 51

Based on the results obtained, it was determined that theoligonucleotide set #1 had a better specificity than set #9. Thisoligonucleotide set had less cross-reactivity to E. coli and was used asone of the oligonucleotide systems for further study.

The other oligonucleotide set used for further study was set #2. Thestructural basis for choosing these two oligonucleotide sets was basedon the combination of enough mismatches to discriminate Salmonella fromother enteric bacteria and enough matches to detect all Salmonellasubspecies. This would allow the amplification system to achieve therequired specificity and sensitivity. The two preferred oligonucleotidesets are shown in Table 2.

Confirmatory testing was performed on both oligonucleotide sets. For set#1, using the new concentrations of T7 (15 pmol), primer (15 pmol) andBlocker (5 pmol) for analyte, and T7 (2 pmol) and primer (2 pmol) forIC, there was a significant improvement in both TTimes (at least 10-15min earlier at 1E+5 -1E+4 S. Enteritidis target copies) and curve shape(standing up and tight). For set #2, using the new concentrations of T7(15 pmol) and primer (15 pmol) for analyte, and T7 (2 pmol) and primer(2 pmol) for IC, there was a significant improvement in both TTimes (atleast 10-17 min earlier at 1E+5-1E+4 S. Enteritidis target copies) andcurve shape (standing up and tight).

Example 5 Evaluation of Target Capture Integration and Internal Control(IC) Integration

Seven Salmonella 23S target capture oligonucleotides (SEQ ID NOs: 71-77were tested using two sets of amplification oligonucleotides: set #7 andset #9. The target capture procedure was performed on varying amounts ofS. Enteritidis GP60 rRNAs and against 1E+7 copies of E. coli GP88 rRNAs.Two potential useful target capture oligonucleotide (TCO) sequences wereidentified (SEQ ID NOs: 71 and 74). Overall, the TTime observed wasabout 8 to 10 min later than in a pure system. Target captureoligonucleotide of SEQ ID NO: 74 was chosen for use in all subsequentexperiments.

The method of Target Capture with Kingfisher 96 is summarized in Table12. Amplification and Enzyme reagents were reconstituted. A wash platewas prepared by filling a KF200 plate with 200 μL/well of wash solution.An amp plate was prepared by filling another KF200 plate with 100μL/well of amplification reagent. Both the amp and wash plates werecovered until used. A sample plate was prepared by adding 50 μL TCR/wellinto a 2-mL, deep-well 96 plate (Axygen). The target was diluted to therequired concentrations in 10 μL lysis solution. One ml of lysissolution was added to each well of the sample plate. With a repeatpipettor, 10 μL of target solution was added to the appropriate deepwells. A deep-well tip-comb was placed in the sample plate. The coversfor the wash and amp plates were removed. The KF96 protocol was startedand all three plates were placed on the KF96 instrument. The amp platewas placed in position 4, the wash plate in position 3, and the sample(deep-well plate) in position 1. Position 2 in the KF96 instrument wasleft empty. Once the plates were loaded, the KF96 instrument began thetarget capture step. When the KF96 run was completed, the plates wereremoved. From the amp plate, 30 μL from each well were removed using amulti-channel pipettor and transferred to an MJ 96-well PCR plate.

TABLE 12 Kingfisher 96 Program Step Step Position Description ActionBeginning Mix End 1 1 Capture Heat 5 min-85° C. Very slow No action 2 1Capture Heat 15 min-65° C. Very slow No action 3 2 Cool Heat 30 min-25°C. (table No action No action rotated to empty position) 4 1 Mix priorto Mix No action 1 min-Very slow Collect beads- collect/collect count 20Sample 1 5 3 Release to Wash Wash Release 30 s Slow 30 s Slow No action6 1 Capture Wash Release 30 s Very 30 s Very Slow Collect beads- Sample2 Slow (mix only) count 20 7 3 Release to Wash Release 30 s Slow 30 sSlow Collect beads- Wash 2 count 20 8 4 Capture and Wash Release 30 sSlow 30 s Slow No action release into Amp Soln

An Internal Control (IC) was integrated into the Salmonella prototypeassay with target capture. This set of IC oligonucleotides performedwell for the Salmonella system with average TTimes in the 19-20 minrange and curves that were tight, sharp and standing up. With the ICintegration, the sensitivity of the Salmonella assay dropped by about10-fold, although it did not seem to affect specificity to other entericbacteria.

TABLE 13 Oligonucleotide Components used for the Complete System usingOligonucleotide Set #2 with IC system Component Salmonella T7 ProviderSEQ ID NO: 26 at 5 pmol Blocker SEQ ID NO: 59 at 0.5 pmol Primer SEQ IDNO: 49 at 10 pmol Torch SEQ ID NO: 66 at 8 pmol Target Capture SEQ IDNO: 74 at 5 pmol Target reference S. Enteritidis GP60 rRNA

Example 6 Sensitivity, Specificity, Interference, Limit of Detection,Cross-Reactivity, and Time to Results

Stage I

Sensitivity

Salmonella Enteritidis, ATCC 13076, was assayed at 1E+5 copies/reaction.Lysis buffer was used as the negative control. Twenty positives (10⁵copies of rRNA/assay) were tested using the KingFisher 96 instrument fortarget capture and the PTI reader for detection. Twenty negatives (lysisbuffer) were used as control. The input for target capture was 1 mL, theoutput for target capture was 100 μL of which 30 μL was used in theamplification. The positive criterion was 1,000 RFU. Nineteen of 20replicates were to be detected with >95% positivity rate. If less than19 replicates were positive after an initial round of testing, 40additional replicates were to be tested. Testing for Stage I-Sensitivityyielded a 100% rate of positivity for Salmonella Enteritidis at 1E+5copies/reaction and 0% false positivity at 0 copies.

Specificity

Organisms that were closely related to the target organism but weregenotypically distinct by rRNA analysis were chosen as negatives. Eightchallenge organisms were tested at 1E+7 copies/rxn using theKingfisher96 instrument for target capture, the Eppendorf thermomixerfor annealing of primers and enzyme addition, and the PTI reader fordetection. Twenty reactions of all challenge organisms (8) were testedwith one replicate of each reaction amplified (10⁵ copies of rRNA, ˜100CFU/assay). S. Enteritidis, ATCC 13076, was used as a positive controlat 1E+5 copies/rxn and lysis solution used as a negative control. Thepositive criterion was 1,000 RFU. Less than or equal to 8 of 160reactions were to meet the goal (to discriminate and not detect 10⁵copies of non-target rRNA) of ≤5% combined false positivity rate. Thedispersion of any false positives across the 8 organisms was to beconsidered. Organisms with clustered false positivity ≥4 were to beretested and investigated further. Stage I-Specificity testing showed 0%positivity against any of the challenge organisms tested and 100%positivity with the positive control.

Interference

The goal was repeatable detection of rRNA approximately equivalent to10-100 CFUs rRNA spiked into a volume of lysis buffer expected to beobtained from the sample concentration device. Testing was to includelow copy numbers of desired rRNA and 10⁷ copies rRNA (˜10,000 CFU)nearest neighbor organisms. S. Enteritidis, ATCC 13076, was used as thebaseline target and was tested at 1E+5 copies rRNA/reaction(approximately 100 CFUs). Eight challenge organisms were spiked into thesamples at a concentration of 0 (lysis solution only) or 1E+7 copies(approximately 10,000 CFU). Assays were performed using the Kingfisher96 and the PTI reader. All conditions were tested in replicates of 12with a positive criterion of 1,000 RFU. Results were to report thereproducibility of positivity in the presence of the nearest neighbororganisms. The dispersion of interference across the organisms testedwas to be considered. Organisms exhibiting interference were to beretested and investigated further. Stage I-Interference testing showed100% positivity in all challenge samples and positive controls.

Microbial Flora Determination

Twenty poultry rinses were analyzed at Gen-Probe to provide an estimateof the normal flora associated with poultry rinse. Eighteen of 20 rinseswere part of one batch that was received from a source outside ofGen-Probe. The other two samples were derived at Gen-Probe from chickenspurchased at 2 local grocery stores. Dilution plating for total aerobiccount on TSA plates was conducted. Dilutions of 1E+1 through 1E+4 ofeach poultry rinsate sample were prepared in 1×phosphate bufferedsaline. 100 μL of undiluted, 1E+1 through 1E+4 rinsate dilutions wereplated on tryptic soy agar (TSA) plates.

Colony counts were performed after the plates had been incubated at 30°C. and 35-37° C. for 24-48 hours. Colonies representing differentmorphologies were sent to PACE Analytical Life Sciences (Minneapolis,Minn.) for identification by RiboPrinter® microbial characterizationsystem. In addition to TSA counts and riboprinting, samples wereenriched in buffered peptone water (BPW) followed by selectiveenrichment with either TT broth or mRSV broth (semi-solid). Samples fromthe selective enrichment were plated on both BGS and XLT4 agar platesfor further selectivity. BIOLOG identification and Gram stain/oxidasetesting were performed on representative colonies. Results showed normalflora in the poultry rinse.

For Salmonella selection, 90 ml buffered-peptone water (BPW) wasinoculated with 10 ml of poultry rinsate and enriched at 35° C. for 24hours. Ten ml of mRSV (modified Rappaport-Vassiliadis-Bouillion) brothwas inoculated with 100 μL of enriched sample and incubated at 42° C.,shaking, for 24 hours. Ten ml of TT broth (Hajna) was inoculated with500 μL of enriched sample and incubated aat 42° C., shaking, for 24hours. Ten μL samples from both selective media (mRSV and TT) wereplated on both XLT4 (xylose lysine tetrathionate) and BGS (brilliantgreen selenite) agar plates. The inoculated plates were incubated at 35°C. and examined at 24 and 48 hours. Selected colonies from the XLT4 andBGS plates were plated on opposite media. For example, if a colony waschosen from the XLT4 plate, it was plated on BGS media, and visa versa.Selected colonies were plated on TSA, from which BIOLOG identificationand Gram stain/oxidase testing were performed to confirm theidentification of the microorganism.

Glycerol stocks of selected colonies were made and sent to PACEAnalytical for riboprinting for confirmation of microorganism identity.

Stage II

Stage II performance testing evaluated the preliminary amplificationassay in Buffered-Peptone Water (BPW). The evaluation used pure culturelysates. Sample preparation device was not included. All positivecontrols (at 1E+5 copies/assay) used the purified RNA isolated from S.enterica ssp. enterica sv. Enteritidis ATCC 13076 and negative controlwas lysis solution:BPW (7:3). Three hundred μL BPW and 700 μL lysisbuffer (with or without sample) were used to make a 1 mL input fortarget capture. The input for target capture was 1 mL, the output fortarget capture was 100 μL of which 30 μL was used in the amplification.

Sensitivity

S. enterica ssp. enterica sv. Choleraesuis (ATCC 10708), S. entericassp. enterica sv. Typhi (ATCC 19430) and S. enterica ssp. enterica sv.Typhimurium (ATCC 13311) were tested at a level of 1E4-5E4 copiesRNA/assay (approximately 10-50 CFU). All three species and negativecontrol (unspiked BPW) were tested in replicates of 20. Target capturewas performed on the Kingfisher 96 instrument, with enzyme addition onthe Eppendorf thermomixer, followed by detection on the PTI reader. Thepositive criterion parameter for the sensitivity was 1,000 RFU. Nineteenof the 20 replicates were to be positive. If less than 19 of 20 werepositive, further testing of an additional 40 replicates was required.All organisms tested for sensitivity passed the Stage II requirement.

Limit of Detection

The goal was repeatable detection of 10³-10⁴ equivalent copies rRNA(˜1-10 CFU) per assay input volume. Repeatable detection was defined as≥95% positivity. S. enterica ssp. enterica sv. Choleraesuis (ATCC10708), S. enterica ssp. enterica sv. Typhi (ATCC 19430), S. entericassp. enterica sv. Typhimurium (ATCC 13311), S. enterica ssp. entericasv. Enteritidis (ATCC 13076), S. enterica ssp. enterica sv. Gallinarum(ATCC 9184) and S. enterica ssp. arizonae (ATCC 29933) were tested at alevel of 1E3-1E4 copies RNA/assay (approximately 1-10 CFU). Targetcapture was performed on the Kingfisher 96, enzyme addition on theEppendorf thermomixer, and the detection on the PTI reader. Each specieswas tested in replicates of 20. The lysates were prepared from pureculture target organisms quantitated in CFUs and lysed to providenucleic acid target at a level equivalent to ˜1-10 CFU. The positivecriterion was 1,000 RFU. S. Enteritidis, ATCC 13076, was considered boththe positive control as well as a strain required for testing. For LODtesting, the Positive Control RNA was used at 1E+4 copies/assay. Thecriteria was ≥95% positivity for all of the species tested. If less than19 of 20 were positive, further testing of an additional 40 replicateswas required. All organisms passed the Stage II requirement.

Analytical Testing of Inclusive and Exclusive Species

Twenty-two Inclusive organisms and twenty-two Exclusive organisms weretested at 1E+5 copies/assay (approximately 100 CFU). Testing wasperformed on the Kingfisher 96 instrument for target capture, enzymeaddition on the Eppendorf thermomixer, and the PTI reader for detection.All were tested in replicates of 4 for the Inclusives and replicates of8 for the Exclusives.

For the Inclusives, 3 of 4 replicates were to be positive and, for theExclusives, no more than 1 of 8 replicates were to be positive. If thesecriteria were not met for any organism, testing for that species/strainwas repeated in replicates of 12, where 11 of 12 replicates ofInclusives were to be reactive. The identity of organisms that failedthe inclusivity criterion after retest were to be further investigated.For Exclusives that did not meet retesting criterion [<3/12 positive],cross-reacting organisms were further investigated.

TABLE 14 Positivity Rate for Inclusives Testing ATCC Copies per No. ofNo. of Organism Serovar # Reaction Reactions Replicates PositivesPositivity S. enterica Typhimurium 33062 1E5 4 1 4 100% ssp. enterica S.bongori 43975 1E5 4 1 0  0% S. enterica Harmelen 15783 1E5 4 1 4 100%ssp. houtenae S. enterica Heidelberg 8326 1E5 4 1 4 100% ssp. entericaS. enterica Newport 6962 1E5 4 1 4 100% ssp. enterica S. entericaMuenchen 8388 1E5 4 1 4 100% ssp. enterica S. enterica Typhi 6539 1E5 41 4 100% ssp. enterica S. enterica Saint Paul 9712 1E5 4 1 4 100% ssp.enterica S. enterica Montevideo 8387 1E5 4 1 4 100% ssp. enterica S.enterica Paratyphi A 9150 1E5 4 1 4 100% ssp. enterica S. entericaParatyphi B 10719 1E5 4 1 4 100% ssp. enterica S. enterica Paratyphi C13428 1E5 4 1 4 100% ssp. enterica S. enterica 33952 1E5 4 1 4 100% ssp.arizonae S. enterica 29934 1E5 4 1 4 100% ssp. diarizonae S. entericaTyphimurium 14028 1E5 4 1 4 100% ssp. enterica S. enterica Illinois11646 1E5 4 1 4 100% ssp. enterica S. enterica Hooggraven 15786 1E5 4 14 100% ssp salamae S. enterica Cubana 12007 1E5 4 1 0  0% ssp. entericaS. enterica Rubislaw 10717 1E5 4 1 4 100% ssp. enterica S. entericaPanama 7378 1E5 4 1 4 100% ssp. enterica S. enterica Gallinarum 9184 1E54 1 4 100% ssp. enterica S. enterica Ferlac 43976 1E5 4 1 4 100% ssp.indica Positive* 13076 1E5 12 1 12 100% Negative* NA 0 12 1 0  0% RepeatTesting S. bongori 43975 1E5 12 1 0  0% S. enterica Cubana 12007 1E5 121 12 100% ssp. enterica *Total for all runs

TABLE 15 Positivity Rate for Exclusives Testing Copies Number NumberATCC#/ per of of Organism other Reaction Reactions Replicates PositivesPositivity E. coli 25922 1E5 8 1 0 0% E. vulneris 33833 1E5 8 1 0 0% E.hermannii 55236 1E5 8 1 0 0% E. cloacae 700644 1E5 8 1 0 0% E. aerogenes13048 1E5 8 1 0 0% E. hoshinae 33379 1E5 8 1 0 0% P. mirabilis 29906 1E58 1 0 0% C. brakii 29063 1E5 8 1 0 0% P. fluorescens 13525 1E5 8 1 0 0%S. flexneri 12022 1E5 8 1 0 0% C. freundii 33128 1E5 8 1 0 0% C.koseri/diversus CI495 1E5 8 1 0 0% K. pneumoniae 23357 1E5 8 1 0 0% S.marcescens 13880 1E5 8 1 0 0% L. innocua 33090 1E5 8 1 0 0% E. faecalis33186 1E5 8 1 0 0% C. jejuni 33560 1E5 8 1 0 0% C. coli 43478 1E5 8 1 00% S. pneumoniae 6303 1E5 8 1 0 0% Positive* 13076 1E5 8 1 8 100% Negative* NA 0 8 1 0 0% *Total for all runs

Testing for the Stage II—Analytical Testing of Inclusives (Table 14) andExclusives (Table 15) was considered complete except for the singleminor exception of Salmonella bongori inclusivity. S. bongori was notdetected upon initial testing of 4 replicates (0 pos/4 reps) andretesting of 12 replicates (0 pos/12 reps) (Table 14). For S. bongori,retesting was done on a new lysate tube from the current lot of S.bongori ATCC 43975 after determination of RNA concentration usingGen-Probe's MTC-NI (cat. no. 4573). Testing was also performed on otherstrains of S. bongori, but none was amplified by the current Salmonellaassay. Due to the rare isolation of this organism, detection of S.bongori was not considered a requirement for the assay as S. bongori hasonly been isolated twice out of 36,184 isolates and is not in the top 30isolates per the CDC 2005 Salmonella Annual Summary. For S. Cubanaretesting, a new lysate tube was used from the same lot used in theinitial testing. Upon retesting, S. Cubana (12 pos/12 reps) passed Stage2 acceptable criteria for retesting.

Time-To-Result

The time of each assay run for Stage II was tracked from the timesamples were added to the deep-well plate through the end of the PTIreader protocol. The average time from sample loading to the start ofthe PTI reader was 2 hours, 18 minutes for 96 samples. The PTI readertime was static at 75 minutes. Therefore, the whole assay from start tofinish was 3 hours, 33 minutes on average for 96 samples.

These results indicate that the species-specific detection of Salmonellacan be achieved by the compositions and methods even in the presence ofclosely related organisms, based upon the characteristics of thereal-time TMA data (e.g., the size and shape of RFU curves generatedfrom the real-time TMA reactions).

Example 7 Food Testing of Spiked Ground Beef and Ice Cream

To test the functionality of the prototype Salmonella assay with reallife samples, ground beef and ice cream were purchased from a localsupermarket, spiked with a known quantity of S. Enteritidis GP60 andgrown in buffered peptone water in a Stomacher® sampling bag. At varioustime points, samples were removed and processed for colony count usingXLD agar selective medium and for real-time TMA using the prototypeSalmonella assay. The various steps followed in this study are describedbelow. A McFarland 1 of S. Enteritidis GP60 was made. CFU countconfirmation in TSA plates (made dilution to 1E+6 in sterile PBS) wasperformed. Twenty-five grams of food was weighed and aseptically placedinto a Stomacher bag. Twenty CFU were inoculated directly to 225 mL ofBuffered Peptone Water. The spiked media was poured into thefood-containing Stomacher bag and processed for 2 minutes at 200 rpm.The sample was incubated at 35° C. A 1-mL aliquot was removed (1 aliquotfor use in plate count) at times 0, 4, 6, 8, and 24 hours. The samplewas plated for CFU counts on selective agar, XLD plates at 3 dilutions,1 plate/dilution and incubated at 35° C. The remaining five aliquotssampled during a 24-hour period were spun at 12,000×g for 30 seconds.The supernatant was removed and 500 μL of a 50 mM succinate buffer (0.6M LiCl, 1% LiLS, pH 4.8) was added to the pellet which was then vortexedvigorously for 20 seconds. The sample was heated at >95° C. at least 15minutes. It was then spun at 12,000×g for 1 minute. The supernatant wastransferred to a new labeled tube. Samples were frozen at −70° C. Foodcontrols included: 2 positive and 2 negative for ground beef, 2 positiveand 2 negative for plain vanilla icecream, and 2 positive and 1 negativefor the media only pure system.

Salmonella CFU Timing and Plate Counts

Using an inoculum of around 12 CFU per 225 ml of media, the spikedSalmonella in ground beef grew to around 280 CFU/ml after 4 h ofincubation in buffered peptone water (BPW). In spiked ice cream, 20CFU/ml were observed after 6 h of incubation in BPW. The ground beef wassubstantially more contaminated than ice cream with other entericbacteria and had over 1,000 CFU/ml after 4 h of incubation. The spikedmedia without any food sample had around 20 CFU/ml after 4 h ofincubation. By 24 h, all spiked and unspiked food samples in BPWhad >1.5E+7 CFU/ml. All negative unspiked media controls did not showany growth. These results corroborate the data obtained from real-timeTMA with regards to CFU timing and early emergence of a positivedetectable signal using real-time TMA.

For food samples that were spiked with 12 CFU of Salmonella per 225 mlBPW, a positive RFU signal for Salmonella was observed after 4 h ofincubation in either ground beef or ice cream. The unspiked ground beefcontrol run produced positive signals (due to indigenous microbialcontamination) after 8 h of incubation and unspiked ice cream controlrun showed positive signal after 24 h incubation. In both unspiked foodsamples, the positive signals emerged very late (>40 min). In theunspiked ground beef sample, these false-positive signals may have becaused by cross-reacting organisms (most probably other entericbacteria) at a very high nucleic acid load (e.g. >6E+8 copiesrRNA/reaction in this sample). In the unspiked ice cream sample, theapparent positive signals were derived from indigenous or contaminatingorganisms that did not grow in the XLD selective medium, but grew inBPW. The positive control (positive spiked media) was positive at 4-h to24-h points. The negative control (negative unspiked media) remainednegative throughout the whole run. These data indicate that real-timeTMA can be used to detect a very low level of Salmonella contaminationwith a minimum of 4 h pre-enrichment in BPW and that the whole processdid not require complex sample processing steps, except for two brief(<1 minute) centrifugation steps.

Assay Summary

Amplification and detection oligonucleotides targeting two regions ofSalmonella nucleic acid, a “450 region” corresponding to from about 380to about 630 nucleotide base positions of E. coli 16S rRNA and a “350region” corresponding to from about 150 to about 425 nucleotide basepositions of E. coli 23s rRNA, were designed and synthesized forevaluation. Designs in the 16S-450 region did not yield goodoligonucleotide candidates for a Salmonella genus assay. Theoligonucleotides cross-reacted with Citrobacter and Enterobacter.

Assay specificity and sensitivity were evaluated using lysed bacterialpellets. CFU and rRNA target levels of the bacterial pellets wereestimated by plating and by a direct DNA probe assay.

The Salmonella assay was 100% sensitive to 22 strains of Salmonellaincluding 6 different subspecies and 16 serotypes of S. enterica ssp.One exception was the S. bongori, which is genotypically more similar toE. coli in the target region than other Salmonella species. TheSalmonella assay was 100% specific against 22 non-Salmonella organismsat 1E+5 copies/assay.

Two food matrices, ice cream and ground beef (25 g), were inoculatedwith S. Enteritidis (−20 CFU) and processed through a Stomacher devicein broth. Plating and the real-time TMA were monitored over a 24-hourtime course. The real-time TMA system utilized two fluorescent probes,one specific for the analyte, one specific for an internal control. Theresults were analyzed based on fluorescence emergence curves. Thereal-time TMA assay was run in less than four hours, reducing the timeneeded for testing in food facilities from days to hours. Resultsindicated that low level Salmonella contamination could be detectedwithin 8 hours, which included 4 hours of pre-enrichment in anon-selective medium follow ˜8000 copies (˜8 CFU/gram food).Interference from high nucleic acid load started at 1.8E+10 copies(˜1.8E+7 CFU)/gram food after 8 h growth.

In summary, real-time TMA technology was suitable for rapid, highlysensitive detection of food-borne pathogens. The assay had a sensitivityof 1E+4 rRNA copies/assay (approximately 10 CFU) for the desiredspecies, Salmonella enterica ssp. enterica sv. EnteritidisGP60/ATCC13076, while excluding various nearest neighbors andpotentially co-contaminating flora at 1E+7 rRNA copies/assay(approximately 10,000 CFU). Utilizing magnetic particle target capturetechnology, interference from ground beef and ice cream samples was notobserved. The data demonstrated a rapid test format that allowedscreening of food samples within a single 8-hour workshift forSalmonella with an improved enrichment protocol.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

The invention claimed is:
 1. A detection oligonucleotide comprising a nucleotide sequence consisting essentially of the sequence of SEQ ID NO: 66, 67, 68, 69, or
 70. 2. The detection oligonucleotide of claim 1, wherein the oligonucleotide is fluorescently labeled.
 3. The detection oligonucleotide of claim 2, wherein the oligonucleotide comprises a quencher.
 4. The detection oligonucleotide of claim 1, wherein the oligonucleotide is a molecular torch oligonucleotide.
 5. A kit comprising a plurality of reagents for detecting a Salmonella target nucleic acid, wherein the plurality of reagents comprises the detection oligonucleotide of claim
 1. 6. The kit of claim 5, wherein the detection oligonucleotide is fluorescently labeled.
 7. The kit of claim 5, wherein the plurality of reagents further comprises one or more amplification oligonucleotides for amplifying the Salmonella target nucleic acid.
 8. The kit of claim 7, wherein the Salmonella target nucleic acid is an amplicon that comprises a sequence corresponding to bases from about 164 to about 209 of SEQ ID NO:
 150. 9. The kit of claim 7, wherein the one or more amplification oligonucleotides for amplifying the Salmonella target nucleic acid comprises a promoter oligonucleotide for isothermally amplifying the Salmonella target nucleic acid.
 10. A nucleic acid complex comprising the detection oligonucleotide of claim 1 hybridized to a Salmonella target nucleic acid.
 11. The nucleic acid complex of claim 10, wherein the detection oligonucleotide is fluorescently labeled.
 12. A composition comprising the detection oligonucleotide of claim 1 and one or more amplification and/or capture oligonucleotides for amplifying and/or capturing a Salmonella target nucleic acid.
 13. The composition of claim 12, wherein the detection oligonucleotide is fluorescently labeled.
 14. The composition of claim 12, further comprising one or more amplification oligonucleotides for amplifying a Salmonella target nucleic acid.
 15. The composition of claim 12, further comprising an RNA polymerase.
 16. A method for detecting Salmonella in a sample, said method comprising a detecting step of detecting the presence or absence of a Salmonella target nucleic acid using the detection oligonucleotide of claim
 1. 17. The method of claim 16, wherein the detection oligonucleotide is fluorescently labeled.
 18. The method of claim 17, wherein the detection oligonucleotide further comprises a quencher.
 19. The method of claim 16, wherein the method further comprises amplifying the Salmonella target nucleic acid using one or more amplification oligonucleotides.
 20. The method of claim 16, wherein the Salmonella target nucleic acid is an amplicon that comprises a sequence corresponding to bases from about 164 to about 209 of SEQ ID NO:
 150. 